Polynucleotide compositions

ABSTRACT

Compositions for stabilizing polynucleic acids and increasing the ability of polynucleic acids to cross cell membranes and act in the interior of a cell. In one aspect, the invention provides a polynucleotide complex between a polynucleotide and certain polyether block copolymers. The polynucleotide complex can further include a polycationic polymer, as well as suitable targeting molecules and surfactants. The invention also provides a polynucleotide complex between a polynucleotide and a block copolymer comprising a polyether block and a polycation block.

[0001] This is a continuation-in-part of Ser. No. 08/912,968, filed Aug.1, 1998, which in turn is a continuation-in-part of Ser. No. 08/342,209,filed Nov. 18, 1994, now U.S. Pat. No. 5,656,611.

FIELD OF THE INVENTION

[0002] The present invention relates to compositions of poly(nucleicacid) polymers such as RNA or DNA polymers and polycations that areassociated, either covalently or noncovalently, with block copolymers ofalkylethers. The complexes are well suited for use as vehicles fordelivering nucleic acid into cells.

BACKGROUND OF THE INVENTION

[0003] The use of antisense poly(nucleic acids) to treat geneticdiseases, cell mutations (including cancer causing or enhancingmutations) and viral infections has gained widespread attention. Thistreatment tool is believed to operate, in one aspect, by binding to“sense” strands of mRNA encoding a protein believed to be involved incausing the disease state sought to be treated, thereby stopping orinhibiting the translation of the mRNA into the unwanted protein. Inanother aspect, genomic DNA is targeted for binding by the antisensepolynucleotide (forming a triple helix), for instance, to inhibittranscription. See Helene, Anti-Cancer Drug Design, 6:569 (1991). Oncethe sequence of the mRNA sought to be bound is known, an antisensemolecule can be designed that binds the sense strand by the Watson-Crickbase-pairing rules, forming a duplex structure analogous to the DNAdouble helix. Gene Regulation: Biology of Antisense RNA and DNA, Eriksonand lxzant, eds., Raven Press, New York, 1991; Helene, Anti-Cancer DrugDesign, 6:569 (1991); Crooke, Anti-Cancer Drug Design, 6:609 (1991). Aserious barrier to fully exploiting this technology is the problem ofefficiently introducing into cells a sufficient number of antisensemolecules to effectively interfere with the translation of the targetedmRNA or the function of DNA.

[0004] One method that has been employed to overcome this problem is tocovalently modify the 5′ or the 3′ end of the antisense polynucleic acidmolecule with hydrophobic substituents. These modified nucleic acidsgenerally gain access to the cells interior with greater efficiency.See, for example, Kabanov et al., FEBS Lett., 259:327 (1990); Boutorinet al., FEBS Lett., 23:1382-1390, 1989; Shea et al, Nucleic Acids Res.,18:3777-3783 (1990). Additionally, the phosphate backbone of theantisense molecules has been modified to remove the negative charge (seefor example, Agris et al., Biochemistry, 25:6268 (1986); Cazenave andHelene in Antisense Nucleic Acids and Proteins: Fundamentals andApplications, Mol and Van der Krol, eds., p. 47 et seq., Marcel Dekker,New York, (1991) or the purine or pyrimidine bases have been modified(see, for example, Antisense Nucleic Acids and Proteins: Fundamentalsand Applications, Mol and Van der Krol, eds., p. 47 et seq., MarcelDekker, New York (1991); Milligan et al. in Gene Therapy For NeoplasticDiseases, Huber and Laso, eds., p. 228 et seq., New York Academy ofSciences, New York (1994). Other attempts to overcome the cellpenetration barrier include incorporating the antisense poly(nucleicacid) sequence into an expression vector that can be inserted into thecell in low copy number, but which, when in the cell, can direct, thecellular machinery to synthesize more substantial amounts of antisensepolynucleic molecules. See, for example, Farhood et al., Ann. N.Y. Acad.Sci., 716:23 (1994). This strategy includes the use of recombinantviruses that have an expression site into which the antisense sequencehas been incorporated. See, e.g., Boris-Lawrie and Temin, Ann. N.Y.Acad. Sci., 716:59 (1994). Others have tried to increase membranepermeability by neutralizing the negative charges on antisense moleculesor other nucleic acid molecules with polycations. See, e.g. Kabanov etal., Soviet Scientific Reviews, Vol. 11, Part 2 (1992); 30 Kabanov etal., Bioconjugate Chemistry 4:448 (1993); Wu and Wu, Biochemistry,27:887-892 (1988); Behr et al., Proc. Natl. Acad Sci U.S.A. 86:6982-6986(1989). There have been problems with systemically administeringpoylnucleotides due to rapid clearance degradation and lowbioavailability. In some cases it would be desirable to targetpolynucleotide molecules to a specific site in the body to specifictarget cells. Also, due to poor or low transport across biologicalbarriers (such as the blood-brain barrier) the transport ofpolynucleotides to targets across this barrier is decreased orimpossible. Additionally, the problems with low oral or rectalbioavailability dramatically hinders the administration of suchpolynucleotides (including oligonucleotides).

[0005] Of course, antisense polynucleic acid molecules are not the onlytype of polynucleic acid molecules that can usefully be made morepermeable to cellular membranes. To make recombinant protein expressionsystems, the expression-directing nucleic acid must be transportedacross the membrane and into the eukaryotic or prokaryotic cell thatwill produce the desired protein. For gene therapy, medical workers tryto incorporate, into one or more cell types of an organism, a DNA vectorcapable of directing the synthesis of a protein missing from the cell oruseful to the cell or organism when expressed in greater amounts. Themethods for introducing DNA to cause a cell to produce a new protein,ribozyme or a greater amount of a protein or ribozyme are called“transfection” methods. See, generally, Neoplastic Diseases, Huber andLazo, eds., New York Academy of Science, New York (1994); Feigner, Adv.Drug Deliv. Rev., 5:163 (1990); McLachlin, et al., Progr. Nucl. AcidsRes. Mol. Biol., 38:91 (1990); Karlsson, S. Blood, 78:2481 (1991);Einerhand and Valerio, Curr. Top. Microbiol. Immunol, 177:217-235(1992); Makdisi et al., Prog. Liver Dis., 10:1 (1992); Litzinger andHuang, Biochim. Biophys. Acta, 11, 13:201 (1992); Morsy et al.,J.A.M.A., 270:2338 (1993); Dorudi et al., British J. Surgery, 80:566(1993).

[0006] A number of the above-discussed methods of enhancing cellpenetration by antisense nucleic acid are generally applicable methodsof incorporating a variety of poly(nucleic acids) into cells. Othergeneral methods include calcium phosphate precipitation of nucleic acidand incubation with the target cells (Graham and Van der Eb, Virology,52:456, 1983), co-incubation of nucleic acid, DEAE-dextran and cells(Sompayrac and Danna, Proc. Natl. Acad. Sci., 12:7575, 1981),electroporation of cells in the presence of nucleic acid (Pofter et al.,Proc. Natl. Acad. Sci., 81:7161-7165, 1984), incorporating nucleic acidinto virus coats to create transfection vehicles (Gitman et al., Proc.Natl. Acad. Sci. U.S.A., 82:7309-7313, 1985) and incubating cells withnucleic acid incorporated into liposomes (Wang and Huang, Proc. Natl.Acad. Sci., 84:7851-7855, 1987).

[0007] Another problem in delivering nucleic acid to a cell is theextreme sensitivity of nucleic acids, particularly ribonucleic acids, tonuclease activity. This problem has been particularly germane to effortsto use ribonucleic acids as anti-sense oligonucleotides. Accordingly,methods of protecting nucleic acid from nuclease activity are desirable.

SUMMARY OF THE INVENTION

[0008] The invention relates to compositions of poly(nucleic acid)polymers such as RNA or DNA polymers and polycations that areassociated, either covalently or noncovalently, with block copolymers ofalkylethers. In a preferred embodiment, the poly(nucleic acids) arecomplexed with a polycation. The nucleic acid is stabilized by thecomplex and, in the complex, has increased permeability across cellmembranes. Accordingly, the complexes are well suited for use asvehicles for delivering nucleic acid into cells.

DETAILED DESCRIPTION OF THE INVENTION

[0009] The invention thus relates to compositions of poly(nucleic acid)polymers such as RNA or DNA polymers, and polycations that areassociated (either covalently or noncovalently) with cationic blockcopolymers.

[0010] Structure of Block Copolymers

[0011] Block copolymers are most simply defined as conjugates of atleast two different polymer segments (Tirrel, M. In: Interactions ofSurfactants with Polymers and Proteins. Goddard E. D. andAnanthapadmanabhan, K. P. (eds.), CRC Press, Boca Raton, Ann Arbor,London, Tokyo, pp. 59-122, 1992). Some block copolymer architectures arepresented below.

Segmented Copolymer Architectures (Circles Indicate Joints of PolymerSegments)

[0012]

[0013] The simplest block copolymer architecture contains two segmentsjoined at their termini to give an A-B type diblock. Consequentconjugation of more than two segments by their termini yields A-B-A typetriblock, A-B-A-B- type multiblock, or even multisegment A-B-C-architectures. If a main chain in the block copolymer can be defined inwhich one or several repeating units are linked to different polymersegments, then the copolymer has a graft architecture of, e.g., anA(B)_(n) type. More complex architectures include for example (AB)_(n)or A_(n)B_(m) starblocks which have more than two polymer segmentslinked to a single center.

[0014] In accordance with the present invention, all of thesearchitectures can be useful for polynucleotide delivery, provided thatthey contain (a) at least one polycationic segment that will bind apolynucleotide (“binding segment”) and (b) at least one water solublesegment that will solubilize the complex formed between the blockcopolymer and polynucleotide (“solubilizing segment”).

[0015] In accordance with the present invention, binding andsolubilizing segments can be, independently of each other, linearpolymers, randomly branched polymers, block copolymers, graftcopolymers, star polymer, star block copolymer, dendrimers or have otherarchitecture including but not limited to combinations of the abovelisted structures. For the purposes of the current invention all thesestructures are collectively called herein “block copolymers”.

[0016] The degree of polymerization of the binding and solubilizingsegments is between about 3 and about 10,000. More preferably, thedegree of polymerization is between about 5 and about 2,000, still morepreferably, between about 20 and about 1,000. The molecular weights ofthe binding and solubilizing segments is between about 600 and about500,000. More preferably, the molecular weights are between about 1000and about 100,000, still more preferably, between about 2000 and about50,000.

[0017] Formulas XVIII-XXIII of the instant invention are diblocks andtriblocks. At the same time, conjugation of polycation segments to theends of polyether of formula XVII yields starblocks (e.g., (ABC)₄ type).In addition, the polyspermine of examples 13-15 (below) are branched.Modification of such a polycation with poly(ethylene oxide) yields amixture of grafted block copolymers and starblocks.

[0018] In one embodiment, the poly(nucleic acids) are complexed with apolycation. The nucleic acid is stabilized by the complex and, in thecomplex, has increased permeability across cell membranes. Accordingly,the complexes are well suited for use as vehicles for delivering nucleicacid into cells.

[0019] In a preferred first embodiment, the block copolymer is selectedfrom the group consisting of polymers of formulas: A-B-A′, A-B, B-A-B′,or L(R′)(R²) (R³) (R⁴) (I) (II) (III) (IV)

[0020] wherein A and A′ are A-type linear polymeric segments, B and B′are B-type linear polymeric segments, and R¹, R², R³ and R⁴ are eitherblock copolymers of formulas (I), (III) or (III) or hydrogen and L is alinking group, with the proviso that no more than two of R¹, R², R³ orR⁴ are hydrogen. In another preferred first embodiment of the invention,the polynucleotide composition further comprises a polycationic polymercomprising a plurality of cationic repeating units.

[0021] The composition provides an efficient vehicle for introducingpolynucleotide into a cell. Accordingly, the invention also relates to amethod of inserting poly(nucleic acid) into cells utilizing the firstembodiment polynucleotide composition of the invention.

[0022] In a second embodiment, the invention provides a polynucleotidecomposition comprising:

[0023] (a) a polynucleotide or derivative thereof;

[0024] (b) a block copolymer having a polyether segment and a polycationsegment, wherein the polyether segment comprises at least an A-typesegment, and the polycation segment comprises a plurality of cationicrepeating units.

[0025] In a preferred second embodiment, the copolymer comprises apolymer of formulas: B-A-R, A-R, A-R-A′ R-A-R′, A-B-R, (V-a) (VI-a)(VII) (VIII-a) (V-b) A-R-B, R-A-B, R-A-B-A, R-A-B-A-R (VI-b) (VIII-b)(VIII-c) (VIII-d)

[0026] wherein A, A′ and B are as described above, wherein R and R′ arepolymeric segments comprising a plurality of cationic repeating units,and wherein each cationic repeating unit in a segment may be the same ordifferent from another unit in the segment. The polymers of thisembodiment can be termed “polyether/polycation” polymers. The R and R′,segments can be termed “R-type” polymeric segments or blocks.

[0027] The polynucleotide composition of the second embodiment providesan efficient vehicle for introducing the polynucleotide into a cell.

[0028] Accordingly, the invention also relates to a method of insertingpoly(nucleic acid) into cells utilizing the second embodimentcomposition of the invention.

[0029] In a third embodiment, the invention provides a polynucleotidecomposition comprising a polynucleotide derivative comprising apolynucleotide segment and a polyether segment attached to one or bothof the polynucleotide 5′ and 3′ ends, wherein the polyether comprises anA-type polyether segment.

[0030] In a preferred third embodiment, the derivative comprises a blockcopolymer of formulas: A-pN, pN-A, A-pN-A', pN-A-B, B-A-pN, A-B-A-pN,pN-A-B-A-pN (IX-a) (X-a) (XI) (XII) (XIII) (XIII-a) (XIII-b) A-pN-R,R-A-pN, A-R-pN, pN-A-R, R-pN-A, pN-R-A (IX-b) (IX-c) (IX-d) (X-b) (X-c)(X-d) B-A-B-pN, pN-B-A-B-pN (X-e) (X-f)

[0031] wherein pN represents a polynucleotide having 5′ to 3′orientation, and A, A′ and B are polyether segments as described above.In another preferred third embodiment, the polynucleotide complexcomprises a polycationic polymer.

[0032] Polymers of formulas (I), (II), (III) or (IV) can also be mixedwith each other or can be mixed either additionally or alternativelywith one or more of the polymers of formula (V-a or b), (VI-a or b),(VII-a or b) and (VIII-a or b) and/or with polynucleotide derivatives offormulas (IX-a, b, c, or d), (X-a, b, c, d, e, or f), (XI), (XII) or(XIII) to provide an efficient vehicle for delivering poly(nucleic acid)to the interior of cells.

[0033] The polynucleotide composition of the third embodiment providesan efficient vehicle for introducing the polynucleotide into a cell.Accordingly, the invention also relates to a method of insertingpoly(nucleic acid) into cells utilizing the third embodiment compositionof the invention.

[0034] A fourth embodiment of the invention relates to apolyetherpolycation copolymer comprising a polymer, a polyether segmentand a polycationic segment comprising a plurality of cationic repeatingunits of formula —NH—R⁰, wherein R⁰ is a straight chain aliphatic groupof 2 to 6 carbon atoms, which may be substituted, wherein said polyethersegments comprise at least one of an A-type of B-type segment. In apreferred fourth embodiment, the polycation polymer comprises a polymeraccording to formulas: B-A-R, A-R, A-R-A′, R-A-R′, (A-B-A)_(n)-R_(m),(V) (VI) (VII) (VIII)

[0035] wherein A, A′ and B are as described above, wherein R and R′ arepolymeric segments comprising a plurality of cationic repeating units offormula —NH—R⁰—, wherein R⁰ is a straight chain aliphatic group havingfrom 2 to 6 carbon atoms, which may be substituted. Each —NH—R⁰—repeating unit in an R-type segment can be the same or different fromanother —NH—R⁰— repeating unit in the segment. A preferred fourthembodiment further comprises a polynucleotide or derivative.

[0036] In a fifth embodiment, the invention provides a polycationicpolymer comprising a plurality of repeating units of formula:

[0037] wherein R⁸ is:

[0038] (1) —(CH₂)_(n)—CH(R¹³)—, wherein n is an integer from 0 to about5, and R¹³ is hydrogen, cycloalkyl having 3-8 carbon atoms, alkyl having1-6 carbon atoms, or (CH₂)_(m)R¹⁴, where m is an integer from 0 to about12 and R¹⁴ is a lipophilic substituent of 6 to 20 carbon atoms;

[0039] (2) a carbocyclic group having 3-8 ring carbon atoms, wherein thegroup can be for example, cycloalkyl or aromatic groups, and which caninclude alkyl having 1-6 carbon atoms, alkoxy having 1-6 carbon atoms,alkylamino having 1-6 carbon atoms, dialkylamino wherein each alkylindependently has 1-6 carbon atoms, amino, sulfonyl, hydroxy, carboxy,fluoro or chloro substituents; or (3) a heterocyclic group, having 3-8ring atoms, which can include heterocycloalkyl or heteroaromatic groups,which can include from 1 to 4 heteroatoms selected from the groupconsisting of oxygen, nitrogen, sulfur and mixtures thereto, and whichcan include alkyl having 1-6 carbon atoms, alkoxy having 1-6 carbonatoms, alkylamino having 1-6 carbon atoms, dialkylamino wherein eachalkyl independently has 1-6 carbon atoms, amino, sulfonyl, hydroxy,carboxy, fluoro or chloro substituents.

[0040] R⁹ is a straight chain aliphatic group of 1 to 12 carbon atoms,and R¹⁰, R¹¹ and R¹² are independently hydrogen, an alkyl group of 1-4carbon atoms. R⁹ preferably comprises 2-10 carbon atoms, morepreferably, 3-8. R¹⁴ preferably includes an intercalating group, whichis preferably an acrydine or ethydium bromide group. The number of suchrepeating units in the polymer is preferably between about 3 and 50,more preferably between about 5 and 20. This, polymer structure can beincorporated into other embodiments of the invention as an R-typesegment or polycationic polymer. The ends of this polymer can bemodified with a lipid substituent. The monomers that are used tosynthesize polymers of this embodiment are suitable for use as themonomers fed to a DNA synthesizer, as described below. Thus, the polymercan be synthesized very specifically. Further, the additionalincorporation of polynucleotide sequences, polyether blocks, andlipophilic substituents can be done using the advanced automationdeveloped for polynucleotide syntheses. The fifth embodiment alsoencompasses this method of synthesizing a polycationic polymer.

[0041] In yet another embodiment, the invention relates to a polymer ofa plurality of covalently bound polymer segments wherein said segmentscomprise (a) at least one polycation segment which segment is a cationichomopolymer, copolymer, or block copolymer comprising at least threeaminoalkylene monomers, said monomers being selected from the groupconsisting of:

[0042] (i) at least one tertiary amino monomer of the formula:

[0043]  and the quaternary salts of said tertiary amino monomer, and(ii) at least one secondary amino monomer of the formula:

[0044]  and the acid addition and quaternary salts of said secondaryamino monomer, in which:

[0045] R¹ is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or aB monomer; each of R² and R³, taken independently of the other, is thesame or different straight or branched chain alkanediyl group of theformula:

—(C_(z)H_(2z))—

[0046]  in which z has a value of from 2 to 8; R⁴ is hydrogen satisfyingone bond of the depicted geminally bonded carbon atom; and R⁵ ishydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; R⁶is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer;R⁷ is a straight or branched chain alkanediyl group of the formula:

—(C_(z)H_(2z))—

[0047]  in which z has a value of from 2 to 8; and R⁸ is hydrogen, alkylof 2 to 8 carbon atoms, an A monomer, or a B monomer; and

[0048] (b) at least one straight or branched chained polyether segmenthaving from about 5 to about 400 monomeric units which is:

[0049] (i) a homopolymer of a first alkyleneoxy monomer —OC_(n)H_(2n)—or

[0050] (ii) a copolymer or block copolymer of said first alkyleneoxymonomer and a second different alkyleneoxy monomer —OC_(m)H_(2m)—, inwhich n has a value of 2 or 3 and m has a value of from 2 to 4.

[0051] The preferred polycationic segments include but are not limitedto polyamines (e.g., spermine, polyspermine, polyethyleneimine,polypropyleneimine, polybutileneimine, polypentyl-eneimine,polyhexyleneimine and copolymers thereof), copolymers of tertiary aminesand secondary amines, partially or completely quaternized amines,polyvinyl pyridine and the quaternary ammonium salts of these polycationsegments. Preferred polycation segments also include aliphatic,heterocyclic or aromatic ionenes (Rembaum et al. Polymer letters, 1968,6;159; Tsutsui, T., In Development in ionic polymers-2, Wilson A. D. andProsser, H. J. (eds.) Applied Science Publishers, London, New York, vol.2, pp. 167-187, 1986). Particularly preferred polycationic segmentsinclude a plurality of cationic repeating units of formula —N—R⁰,wherein R⁰ is a straight chain aliphatic group of 2 to 6 carbon atoms,which may be substituted. Each —N—R⁰— repeating unit in an polycationsegment can be the same or different from another —N—R⁰— repeating unitin the segment.

[0052] The polycationic segments in the copolymers of the invention canbe branched. For example, polyspermine-based copolymers are branched.The cationic segment of these copolymers was synthesized by condensationof 1,4-dibromobutane and N-(3-aminopropyl)-1,3-propanediamine. Thisreaction yields highly branched polymer products with primary,secondary, and tertiary amines.

[0053] An example of branched polycations are products of thecondensation reactions between polyamines containing at least 2 nitrogenatoms and alkyl halides containing at least 2 halide atoms (includingbromide or chloride). In particular, the branched polycations areproduced as a result of polycondensation. An example of this reaction isthe reaction between N-(3-aminiopropyl)-1,3-propanediamine and1,4-dibromobutane, producing polyspermine.

[0054] Another example of a branched polycation is polyethyleneiminerepresented by the formula:

(NHCH₂CH₂)_(x)[N(CH₂CH₂)CH₂CH₂]_(y)

[0055] Additionally, cationic dendrimers, for example, polyamidoaminesor polypropyleneimines of various generations (i.e., molecular weight),(Tomalia et al., Angew. Chem., Int. Ed. Engl. 1990, 29, 138) can be alsoused as polycation segments of block copolymers for gene delivery.

[0056] In yet another embodiment, the invention relates to a polymer ofa plurality of covalently bound polymer segments wherein the segmentscomprise:

[0057] (a) at least one polycation segment which is a cationichomopolymer or copolymer comprising at least three cationic amino acids,or at least three aminoalkylene monomers, the monomers selected from thegroup consisting of:

[0058] (i) at least one tertiary amino monomer of the formula:

[0059]  and the quaternary salts of said tertiary amino monomer, and(ii) at least one secondary amino monomer of the formula:

[0060]  and the acid addition and quaternary salts of said secondaryamino monomer, in which:

[0061] R¹ is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or aB monomer; each of R² and R³, taken independently of the other, is thesame or different straight or branched chain alkanediyl group of theformula:

—(C_(z)H_(2z))—

[0062]  in which z has a value of from 2 to 8; R⁴ is hydrogen satisfyingone bond of the depicted geminally bonded carbon atom; and R⁵ ishydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; R⁶is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer;R⁷is a straight or branched chain alkanediyl group of the formula:

—(C_(z)H_(2z))—

[0063]  in which z has a value of from 2 to 8; and R⁸ is hydrogen, alkylof 2 to 8 carbon atoms, an A monomer, or a B monomer; and

[0064] (b) at least one water-soluble nonionic polymer segment. Thisincludes at least one nonionic polymer segment comprising at least threeof the same or different repeating units containing at least one atomselected from the group consisting of oxygen and nitrogen.

[0065] In this embodiment the polycation serves as the binding segment,while the nonionic polymer serves as a lypohilizing segment.

[0066] The polycation segments preferred in this embodiment are the sameas polycations preferred in the previous embodiments. These preferredpolycation segments include but are not limited to polyamines (e.g,spermine, polyspermine, polyethyleneimine, polypropyleneimine,polybutilene-imine, poolypentyleneimine, polyhexyleneimine andcopolymers thereof), copolymers of tertiary amines and secondary amines,partially or completely quaternized amines, polyvinyl pyridine and thequaternary ammonium salts of said polycation segments.

[0067] It is preferred that nonionic polymer segments comprisewater-soluble polymers, which are nontoxic and nonimunogenic. Thepreferred nonionic polymer segment is at least one water-solublenonionic polymer segment is a homopolymer or copolymer of at least oneof the monomers selected from the group consisting of acrylamide,gycerol, vinylalcohol, vinylpyrrolidone, vinylpyridine, vinylpyridineN-oxide, oxazoline, or a acroylmorpholine, and derivatives thereof. Thisincludes for example polyacrylamides, polygycerols, polyvinylalcohols,polyvinylpyrrolidones, polyvinylpyridine N-oxides, copolymers ofvinylpyridine N-oxide and vinylpyridine, polyoxazolines,polyacroylmorpholines or derivatives thereof. Nonionic segmentscomprising products of polymerization of vinyl monomers are alsopreferred, including but not limiting to the following nonionic polymersegments and derivatives thereof:

[0068] in which m a value of from 3 to about 10,000.

[0069] Included within the scope of the invention are compositionscomprising these polymers and a suitable targeting molecule. Alsoincluded within the scope of the invention are compositions comprisingpolymer, a polynucleotide, and a surfactant. The invention also relatesto copolymers comprising at least one polynucleotide segment and atleast one polyether segment, said polyether segment comprisingoxyethylene and oxypropylene.

[0070] The present compositions can be used in a variety of treatments.For example, the compositions can be used in Gene therapy including genereplacement or excision therapy, and gene addition therapy (B. Huber,Gene therapy for neoplastic diseases; B E Huber and J S Lazo Eds., TheNew York Academy of Sciences, NY, N.Y., 1994, pp. 6-11). Also, antisensetherapy targets genes in the nucleus and/or cytoplasm of the cell,resulting in their inhibition (Stein and Cheng, Science 261:1004, 1993;De Mesmaeker et al. Acc. Chem. Res. 28:366, 1995). Aptamer nucleic aciddrugs target both intra-and extracellular proteins, peptides and smallmolecules. See Ellington and Szostak, Nature (London), 346,818, 1990.Antigen nucleic acid compounds can be used to target duplex DNA in thenucleus. See Helene and Tolume, Biochim, Biophys. Acta 1049:99, 1990.Catalytic polynucleotides target mRNA in the nucleus and/or cytoplasm(Cech, Curr. Opp. Struct. Biol. 2:605, 1992).

[0071] Examples of genes to be replaced, inhibited and/or added include,but are not limited to, adenosine deaminase, tumor necrosis factor, cellgrowth factors, Factor IX, interferons (such as α-, β- andγ-interferon), interleukins (such interleukin 2,4, 6 and 12), HLA-B7,HSV-TK, CFTR, HIV-1, β-2, microglobulin, retroviral genes (such as gag,pol, env, tax, and rex), cytomegalovirus, herpes viral genes (such asherpes simplex virus type I and II genes ICP27/UL54, ICP22/US1,ICP/IE175, protein kinase and exonuclease/UL13, protein kinase/US3,ribonuclease reductase ICP6/UL39, immediate early (IE) mRNAIE3/IE175/ICP4, 1E4/ICP22/US1, IE5/ICP47, IE110, DNA polymerase/UL30,UL13), human multidrug resistance genes (such as mdrl), oncogenes (suchas H-c-ras, c-myb, c-myb, bcl-2, bcr/abl), tumor suppressor gene p53,human MHC genes (such as class 1 MHC), immunoglobulins (such as IgG,IgM, IgE, IgA), hemoglobin α- and β-chains, enzymes (such as carbonicanhydrase, triosephoshate isomerase, GTP-cyclhydrdolase I, phenylalaninehydrolase, sarcosine dehydrogenase, glucocerobrosidase,glucose-6-phosphate dehydrogenase), dysotrophin, fibronectin,apoliprotein E, cystic fibrosis transmembrane conductance protein, c-srcprotein, V(D)J recombination activating protein, immunogenes, peptideand protein antigens (“DNA vaccine”) and the like.

[0072] Genetic diseases can also be treated by the instant compositions.Such diseases include, but are not limited to, rheumatoid arthritis,psoriasis, Crohn's disease, ulcerative colitis, α-thalassemia,β-thalassemia, carbonic anhydrase II deficiency syndrome,triosephosphate isomerase deficiency syndrome,tetrahydrobiopterindeficient hyperphenylalaniemia, classicalphenylketonuria, muscular dystrophy such as Duchenne Muscular Dystrophy,hypersarkosinemia, adenomatous intestinal polyposis, adenosine deaminasedeficiency, malignant melanoma, glucose-6-phosphste dehydrogenasedeficiency syndrome, arteriosclerosis and hypercholesterolemia,Gaucher's disease, cystic fibrosis, osteopetrosis, increased spontaneoustumors, T and B cell immunodeficiency, high cholesterol, arthritisincluding chronic rheumatoid arthritis, glaucoma, alcoholism and thelike.

[0073] The compositions can also be used to treat neoplastic diseasesincluding, but not limited to, breast cancer (e.g., breast, pancreatic,gastric, prostate, colorectal, lung, ovarian), lymphomas (such asHodgkin and non-Hodgkin lymphoma), melanoma and malignant melanoma,advanced cancer hemophilia B, renal cell carcinoma, gliblastoma,astrocytoma, gliomas, AML and CML and the like.

[0074] Additionally, the compositions can be used to treat (i)cardiovascular diseases including but not limited to stroke,cardiomyopathy associated with Duchenne Muscular Dystrophy, myocardialischemia, restenosis and the like, (ii) infectious diseases such asHepatitis, HIV infections and AIDS, Herpes, CMV and associated diseasessuch as CMV renitis, (iii) transplantation related disorders such asrenal transplant rejection and the like, and (iv) are useful in vaccinetherapies and immunization, including but not limited to melanomavaccines, HIV vaccines, malaria, tuberculosis, and the like.

[0075] Target Cells

[0076] Cell targets can be ex vivo and/or in vivo, and include T and Blymphocytes, primary CML, tumor infiltrating lymphocytes, tumor cells,leukemic cells (such as HL-60, ML-3, KG-1 and the like), skinfibroblasts, myoblasts, cells of central nervous system includingprimary neurons, liver cells, carcinoma (such as Bladder carcinoma T24,human colorectal carcinoma Caco-2), melanoma, CD34+ lymphocytes, NKcells, macrophages, hemotopoetic cells, neuroblastoma (such as LAN-5 andthe like), gliomas, lymphomas (such as Burkitt lymphomas ST486), JD38),T-cell hybridomas, muscle cells such as primary smooth muscle, and thelike.

[0077] Filed concurrently with the parent of this application (Nov. 18,1994) was Ser. No. 08/342,079 entitled “POLYMER LINKED BIOLOGICALAGENTS”. The entire disclosure of that application is incorporatedherein by reference.

[0078] The degree of polymerization of the hydrophilic (A-type) segmentsor the hydrophobic (B-type) segments of formulas (I)-(XIII) canpreferably be between about 5 and about 400. More preferably, the degreeof polymerization is between about 5 and about 200, still morepreferably, between about 5 and about 80. The degree of polymerizationof the R-type polycation segments can preferably be between about 2 andabout 300. More preferably, the degree of polymerization is betweenabout 5 and about 180, still more preferably, between about 5 and about60. The degree of polymerization of the polycationic polymer canpreferably be between about 10 and about 10,000. More preferably, thedegree of polymerization is between about 10 and about 1,000, still morepreferably, between about 10 and about 100.

[0079] The repeating units that comprise the segments, for A-type,B-type and R-type segments, will generally have molecular weight betweenabout 30 and about 500, preferably between about 30 and about 100, stillmore preferably between about 30 and about 60. Generally, in each of theA-type or B-type segments, at least about 80% of the linkages betweenrepeating units are ether linkages, preferably, at least about 90% areether linkages, more preferably, at least about 95% are ether linkages.Ether linkages, for the purposes of this application, encompassglycosidic linkages (i.e., sugar linkages). However, in one aspect,simple ether linkages are preferred.

[0080] The polynucleotide component (pN) of formulas (IX) through (XIII)will preferably comprise from about 5 to about 1,000,000 bases, morepreferably about 5 to about 100,000 bases, yet more preferably about 10to about 10,000 bases.

[0081] The polycation segments have several positively ionizable groupsand a net positive charge at physiologic pH. The polyether/polycationpolymers of formulas (V)-(VIII) can also serve as polycationic polymers.Preferably, the polycation segments have at least about 3 positivecharges at physiologic pH, more preferably, at least about 6, still morepreferably, at least about 12. Also preferred are polymers or segmentsthat, at physiologic pH, can present positive charges with a distancebetween the charges of about 2 Å to about 10 Å. The distancesestablished by ethtyleneimine, aminopropylene, aminobutilene,aminopentylene and aminohehhylene repeating units, or by mixtures of atleast two of these groups are most preferred. Preferred are polycationicsegments that utilize (NCH₂CH₂), (NCH₂CH₂CH₂), (NCH₂CH₂CH₂CH₂),(NCH₂CH₂CH₂CH₂CH₂), and (NCH₂CH₂CH₂CH₂CH₂CH₂) repeating units, or amixture thereof.

[0082] Polycation segments having an —N—R⁰— repeating unit are alsopreferred. R⁰ is preferably an ethylene, propylene, butylene, pentylene,or hexylene which can be modified. In a preferred embodiment, in atleast one of the repeating units RW includes a DNA intercalating groupsuch as an ethidium bromide group. Such intercalating groups canincrease the affinity of the polymer for nucleic acid. Preferredsubstitutions on R⁰ include alkyl of 1-6 carbon atoms, hydroxy,hydroxyalkyl, wherein the alkyl has 1-6 carbon atoms, alkoxy having 1-6carbon atoms, an alkyl carbonyl group having 2-7 carbon atoms,alkoxycarbonyl wherein the alkoxy has 1-6 carbon atoms,alkoxycarbonylalkyl wherein the alkoxy and alkyl each independently has1-6 carbon atoms, alkylcarboxyalkyl wherein each alkyl group has 1-6carbon atoms, aminoalkyl wherein the alkyl group has 1-6 carbon atoms,alkylamino or dialkylamino where each alkyl group independently has 1-6carbon atoms, mono- or di-alkylaminoalkyl wherein each alkylindependently has 1-6 carbon atoms, chloro, chloroalkyl wherein thealkyl has from 1-6 carbon atoms, fluoro, fluoroalkyl wherein the alkylhas from 1-6 carbon atoms, cyano, or cyano alkyl wherein the alkyl hasfrom 1-6 carbon atoms or a carboxyl group. More preferably, R⁰ isethylene, propylene or butylene.

[0083] Polymers according to the first embodiment of the invention. areexemplified by the block copolymers having the formulas:

[0084] in which x, y, z, i and j have values from about 5 to about 400,preferably from about 5 to about 200, more preferably from about 5 toabout 80, and wherein for each R¹, R² pair, one is hydrogen and theother is a methyl group.

[0085] Formulas (XIV) through (XVI) are oversimplified in that, inpractice, the orientation of the isopropylene radicals within the Bsegment will be random. This random orientation is indicated in formula(XVII), which is more complete. Suchpoly(oxyethylene)-poly(oxypropylene) compounds have been described bySanton, Am. Perfumer Cosmet., 72(4):54-58 (1958); Schmolka, Loc. cit.82(7):25 (1967); Schick, Non-ionic Surfactants, pp. 300-371 (Dekker, NY,1967). A number of such compounds are commercially available under suchgeneric trade names as “poloxamers”, “pluronics” and “synperonics.”Pluronic polymers within the B-A-B formula are often referred to as“reversed” pluronics, “pluronic R” or “meroxapol”. The “polyoxamine”polymer of formula (XVII) is available from BASF (Wyandotte, Mich.)under the tradename Tetronic™. The order of the polyoxyethylene andpolyoxypropylene segments represented in formula (XVII) can be reversed,creating Tetronic R™, also available from BASF. See, Schmolka, J. Am.Oil Soc., 59:110 (1979). Polyoxypropylene-polyoxyethylene blockcopolymers can also be designed with hydrophilic segments comprising arandom mix of ethylene oxide and propylene oxide repeating units. Tomaintain the hydrophilic character of the segment, ethylene oxide willpredominate. Similarly, the hydrophobic segment can be a mixture ofethylene oxide and propylene oxide repeating units. Such blockcopolymers are available from BASF under the tradename Pluradot™

[0086] The diamine-linked pluronic of formula (XVII) can also be amember of the family of diamine-linked polyoxyethylene-polyoxypropylenepolymers of formula:

[0087] wherein the dashed lines represent symmetrical copies of thepolyether extending off the second nitrogen, R* is an alkylene of 2 to 6carbons, a cycloalkylene of 5 to 8 carbons or phenylene, for R¹ and R²,either (a) both are hydrogen or (b) one is hydrogen and the other ismethyl, for R³ and R⁴ either (a) both are hydrogen or (b) one ishydrogen and the other is methyl, if both of R³ and R⁴ are hydrogen,then one R⁵ and R⁶ is hydrogen and the other is methyl, and if one of R³and R⁴ is methyl, then both of R⁵ and R⁶ are hydrogen.

[0088] Those of ordinary skill in the art will recognize, in light ofthe discussion herein, that even when the practice of the invention isconfined for example, to poly(oxyethylene)-poly(oxypropylene) compounds,the above exemplary formulas are too confining. Thus, the units makingup the first segment need not consist solely of ethylene oxide.Similarly, not all of the B-type segment need not consist solely ofpropylene oxide units. Instead, the segments can incorporate monomersother than those defined in formulas (XIV)-(XVII), so long as theparameters of the first embodiment are maintained. Thus, in the simplestof examples, at least one of the monomers in segment A might besubstituted with a side chain group as previously described.

[0089] In another aspect, the invention relates to a polynucleotidecomplex comprising a block copolymer at least one of formulas(I)-(XIII), wherein the A-type and B-type segments are substantiallymade up of repeating units of formula —O—R⁹, where R⁹ is:

[0090] (1) —(CH₂)_(n)—CH(R⁶), wherein n is an integer from 0 to about 5and R⁶ is hydrogen, cycloalkyl having 3-8 carbon atoms, alkyl having 1-6carbon atoms, phenyl, alkylphenyl wherein the alkyl has 1-6 carbonatoms, hydroxy, hydroxyalkyl, wherein the alkyl has 1-6 carbon atoms,alkoxy having 1-6 carbon atoms, an alkyl carbonyl group having 2-7carbon atoms, alkoxycarbonyl, wherein the alkoxy has 1-6 carbon atoms,alkoxycarbonylalkyl, wherein the alkoxy and alkyl each independently has1-6 carbon atoms, alkylcarboxyalkyl, wherein each alkyl group has 1-6carbon atoms, aminoalkyl wherein the alkyl group has 1-6 carbon atoms,alkylamine or dialkylamino, wherein each alkyl independently has 1-6carbon atoms, mono- or di-alkylaminoalkyl wherein each alkylindependently has 1-6 carbon atoms, chloro, chloroalkyl wherein thealkyl has from 1-6 carbon atoms, fluoro, fluoroalkyl wherein the alkylhas from 1-6 carbon atoms, cyano or cyano alkyl wherein the alkyl hasfrom 1-6 carbon atoms or carboxyl; (2) a carbocyclic group having 3-8ring carbon atoms, wherein the group can be for example, cycloalkyl oraromatic groups, and which can include alkyl having 1-6 carbon atoms,alkoxy having 1-6 carbon atoms, alkylamino having 1-6 carbon atoms,dialkylamino wherein each alkyl independently has 1-6 carbon atoms,amino, sulfonyl, hydroxy, carboxy, fluoro or chloro substitutions, or(3) a heterocyclic group, having 3-8 ring atoms, which can includeheterocycloalkyl or heteroaromatic groups, which can include from 1-4heteroatoms selected from the group consisting of oxygen, nitrogen,sulfur and mixtures thereto, and which can include alkyl having 1-6carbon atoms, alkoxy having 1-6 carbon atoms, alkylamino having 1-6carbon atoms, dialkylamino wherein each alkyl independently has 1-6carbon atoms, amino, sulfonyl, hydroxy, carboxy, fluoro or chlorosubstitutions.

[0091] Preferably, n is an integer from 1 to 3. The carbocyclic orheterocyclic groups comprising R⁵ preferably have from 4-7 ring atoms,more preferably 5-6. Heterocycles preferably include from 1-2heteroatoms, more preferably, the heterocycles have one heteroatom.Preferably, the heterocycle is a carbohydrate or carbohydrate analog.Those of ordinary skill will recognize that the monomers required tomake these polymers are synthetically available. In some cases,polymerization of the monomers will require the use of suitableprotective groups, as will be recognized by those of ordinary skill inthe art. Generally, the A- and B-type segments are at least about 80%comprised of —OR⁵— repeating units, more preferably at least about 90%,yet more preferably at least about 95%.

[0092] In another aspect, the invention relates to a polynucleotidecomplex comprising a block copolymer of one of formulas (I)-(XIII)wherein the A-type and B-type segments consist essentially of repeatingunits of formula —O—R⁵ wherein R⁷ is a C to C alkyl group.

[0093] A wide variety of nucleic acid molecules can be the nucleic acidcomponent of the composition. These include natural and synthetic DNA orRNA molecules and nucleic acid molecules that have been covalentlymodified (to incorporate groups including lipophilic groups,photo-induced crosslinking groups, alkylating groups, organometallicgroups, intercalating groups, lipophilic groups, biotin, fluorescent andradioactive groups, and groups that modify the phosphate backbone). Suchnucleic acid molecules can be, among other things, antisense nucleicacid molecules, gene-encoding DNA (usually including an appropriatepromoter sequence), ribozymes, aptamers, antigen nucleic acids,oligonucleotide α-anomers, ethylphosphotriester analogs,alkylphosphomates, phosphorothionate and phosphorodithionateoligonucleotides, and the like. In fact, the nucleic acid component canbe any nucleic acid that can beneficially be transported into a cellwith greater efficiency, or stabilized from degradative processes, orimproved in its biodistribution after administration to an animal.

[0094] Examples of useful polymers pursuant to formulas (V)-(VIII)include the poly(oxyethylene)-poly-L-lysine) diblock copolymer of thefollowing formula:

[0095] wherein i is an integer of from about 5 to about 100, and j is aninteger from about 4 to about 100. A second example is thepoly(oxyethylene)-poly-(L-alanine-L-lysine) diblock copolymer offormula:

[0096] wherein i is an integer of from about 5 to about 100, and j is aninteger from about 4 about 100. A third example is thepoly(oxyethylene)-poly(propyleneimine/butyleneimine) diblock copolymerof the following formula:

[0097] wherein i is an integer from about 5 about 200 and j is aninteger from 1 to about 10. A fourth example is thepoly(oxyethylene)-poly(N-ethyl-4-vinylpyridinium bromide)(“pOE-pEVP-Br”) of formula:

[0098] wherein i is an integer of from about 5 to about 100 and j is aninteger of from about 10 to about 500. Still another example is thepolymer of formula:

CH₃O—(CH₂CH₂O)_(i)CO[(NH(CH₂)₃)₂NH(CH₂)₄]_(j)—(NH(CH₂)₃)₂—NHCO—O—(CH₂CH₂O)_(k)—CH₃  (XXII)

[0099] wherein i is an integer from about 10 to about 200, j is aninteger from about 1 to about 8, and k is an integer from about 10 toabout 200. Still another example is the polymer of formula:

H—G_(j)—(NH(CH₂)₃)₂—N—NH—CO—O—(CH₂CH₂O)_(i)CO—G_(m)—(NH(CH₂)₃)₂—NH₂  (XXIII)

[0100] wherein “G” comprises —(NH(CH₂)₃)₃—CH₂NH₂—, i and j are asdefined for formula (XVIII), and m is an integer from about 1 to about8.

[0101] The block copolymers utilized in the invention will typically,under certain circumstances, form micelles or micelle-like aggregates offrom about 10 nm to about 100 nm in diameter. Micelles aresupramolecular complexes of certain amphiphilic molecules that form inaqueous solutions due to microphase separation of the nonpolar portionsof the amphiphiles. Micelles form when the concentration of theamphiphile reaches, for a given temperature, a critical micellarconcentration (“CMC”) that is characteristic of the amphiphile. Suchmicelles will generally include from about 10 to about 300 blockcopolymers. By varying the sizes of the hydrophilic and hydrophobicportions of the block copolymers, the tendency of the copolymers to formmicelles at physiological conditions can be varied. The micelles have adense core formed by the water insoluble repeating units of the B blocksand charge-neutralized nucleic acids, and a hydrophilic shell formed bythe A segments. The micelles have translational and rotational freedomin solution, and solutions containing the micelles have low viscositysimilar to water. Micelle formation typically occurs at copolymerconcentrations from about 0.001 to 5% (w/v). Generally, theconcentration of polycationic polymers and polynucleic acid will be lessthan the concentration of copolymers in the polynucleotide compositions,preferably at least about 10-fold less, more preferably at least about50-fold.

[0102] At high concentrations, some of the block copolymers utilized inthe invention will form gels. These gels are viscous systems in whichthe translational and rotational freedom of the copolymer molecules issignificantly constrained by a continuous network of interactions amongcopolymer molecules. In gels, microsegregation of the B segmentrepeating units may or may not occur. To avoid the formation of gels,polymer concentrations (for both block copolymers andpolyether/polycation polymers) will preferably be below about 15% (w/v),more preferably below about 10%, still more preferably below about 5%.In the first embodiment of the invention, it is more preferred that gelsbe avoided.

[0103] When the polynucleotide composition includes cationic components,the cations will associate with the phosphate groups of thepolynucleotide, neutralizing the charge on the phosphate groups andrendering the polynucleotide component more hydrophobic. Theneutralization is preferably supplied by cations on R-type polymericsegments or on polycationic polymers. However, the phosphate charge canalso be neutralized by chemical modification or by association with ahydrophobic cations such as N-[1-(2,3-dioleyloxy)-N,N′-3-methylammoniumchloride]. In aqueous solution, the charge neutralized polynucleotidesare believed to participate in the formation of supramolecular,micelle-like particles, termed “polynucleotide complexes.” Thehydrophobic core or the complex comprises the charge neutralizedpolynucleotides and the B-type segments. The hydrophilic shell comprisesthe A-type segments. The size of the complex will generally vary fromabout 10 nm to about 100 nm in diameter. In some contexts, it ispractical to isolate the complex from unincorporated components. Thiscan be done, for instance, by gel filtration chromatography.

[0104] The ratio of the components of the polynucleotide composition isan important factor in optimizing the effective transmembranepermeability of the polynucleotides in the composition. This ratio canbe identified as ratio Ø, which is the ratio of positively chargedgroups to negatively charged groups in the composition at physiologicalpH. If Ø<1, the complex contains non-neutralized phosphate from thepolynucleotide. The portions of the polynucleotides adjacent to thenon-neutralized charges are believed to be a part of the shell of apolynucleotide complex. Correspondingly, if Ø>1, the polycationicpolymer or R-type segment will have non-neutralized charges, and theun-neutralized portions will fold so that they form a part of the shellof the complex. Generally, Ø will vary from about 0 (where there are nocationic groups) to about 100, preferably Ø will range between about0.01 and about 50, more preferably, between about 0.1 and about 20. Øcan be varied to increase the efficiency of transmembrane transport and,when the composition comprises polynucleotide complexes, to increase thestability of the complex. Variations in Ø can also affect thebiodistribution of the complex after administration to an animal. Theoptimal Ø will depend on, among other things, (1) the context in whichthe polynucleotide composition is being used, (2) the specific polymersand oligonucleotides being used, (3) the cells or tissues targeted, and(4) the mode of administration.

[0105] It will in some circumstances be desirable to incorporate in thepolynucleotide compositions of the current invention, by noncovalentassociation or covalent conjugation, targeting molecules. See forexample, Kabanov et al., J. Controlled Release, 22:141 (1992), thecontents of which are hereby incorporated by reference. The term“targeting molecule” means any molecule, atom, or ion that enhancesbinding, transport, accumulation, residence time, bioavailability ormodifies biological activity of the polynucleotides or thepolynucleotide compositions of the current invention in the body orcell. The targeting molecule will frequently comprise an antibody,fragment of antibody or chimeric antibody molecule typically withspecificity for a certain cell surface antigen. The targeting moleculecan also be, for instance, a hormone having a specific interaction witha cell surface receptor, or a drug having a cell surface receptor. Forexample, glycolipids can serve to target a polysaccharide receptor. Thetargeting molecules can also be, for instance, enzymes, lectins, andpolysaccharides. low molecular mass ligands, such as folic acid andderivatives thereof are also useful in the context of the currentinvention. The targeting molecules can also be polynucleotide,polypeptide, peptidomimetic, carbohydrates including polysaccharides,derivatives thereof or other chemical entities obtained by means ofcombinatorial chemistry and biology. The targeting molecules can be usedto facilitate intracellular transport of the polynucleotide compositionsof the invention, for instance transport to the nucleus, by using, forexample, fusogenic peptides as targeting molecules described bySoukchareun et al., Bioconjugate Chem., 6, 43 (1995), or Arar et al.,Bioconjugate Chem., 6, 43 (1995), caryotypic peptides, or otherbiospecific groups providing site-directed transport into a cell (inparticular, exit from endosomic compartments into cytoplasm, or deliveryto the nucleus).

[0106] Included within the scope of the invention are compositionscomprising the polynucleotide, block copolymer of the current inventionand a suitable targeting molecule. The targeting molecule can becovalently linked to any of the polymer segments of the block copolymersidentified herein (or polynucleotide complexes thereof), includingcationic and nonionic polymer segments. For instance, the targetingmolecule can be linked to the free-terminal or pendant groups of thenonionic segments. Such targeting molecules can be linked to theterminal or pendant —OH end group of the polymer segments, and theterminal or pendant —NH₂ group of the polymer segments, or the terminalor pendant —COOH end group of the polymer segments, or the like.

[0107] It will in some circumstances be desirable to incorporatetargeting molecules through ligand-receptor constructs, in which:

[0108] (i) the ligand molecule is a chemical entity (e.g., a molecule,atom, or ion) capable of specific binding with the receptor molecule;(ii) the receptor molecule is a chemical entity capable of specificbinding to the ligand molecule; or (iii) the ligand molecules, receptormolecules (or both) are incorporated into the block copolymers (orpolynucleotide complexes thereof), targeting molecules, or both. This isdone by noncovalent association or covalent conjugation so that after(i) mixing targeting molecules and block copolymers (or polynucleotidecomplexes thereof) with the ligand and receptor molecules attached tothem, or (ii) by adding either free ligand or receptor (or both) to themixture of targeting molecules and block copolymers (or polynucleotidecomplexes thereof), the targeting molecule becomes attached to the blockcopolymers (or to the polynucleotide complexes thereof) throughligand-receptor binding. Examples of such constructs include constructsusing biotin as the ligand and avidin or streptavidin as the receptor.For example, biotin or a derivative thereof can be covalently linked tothe block copolymers (or polynucleotide complexes thereof) of thecurrent invention and avidin (or streptavidin) can be covalently linkedto the targeting molecule. Alternatively, biotin can be linked to boththe block copolymers (or polynucleotide complexes thereof) and targetingmolecule, and the latter can be linked through avidin, which has fourbiotin-binding centers. Further, additional complex constructscomprising biotin and avidin can be used for incorporating targetingmolecules in polynucleotide compositions of the current invention. Inone particular embodiment, the invention provides for the blockcopolymers (or polynucleotide complexes thereof) with biotin moleculesor derivatives thereof linked to at least one polycation or nonionicpolymer segments or both polycation and nonionic polymer segments. Thoseof ordinary skill in the art will recognize, in light of the discussionherein, that even when the practice of the invention is confined forexample, to biotin-avidin or biotin-streptavidin constructs or thesimilar constructs, there are numerous ways available providing for thedesign of the ligand-receptor constructs with the desiredcharacteristics pursuant to this invention. Such constructs, forexample, can comprise ligands and/or receptors that are polynucleotide,polypeptide, peptidomimetic, carbohydrates including polysaccharides,derivatives thereof or other chemical entities obtained by means ofcombinatorial chemistry and biology.

[0109] The targeting molecules which can be associated with thepolynucleotide compositions of the invention can also have a targetinggroup having affinity for a cellular site and a hydrophobic group. Suchtargeting molecules can provide for the site specific delivery andrecognition in the body. The targeting molecule spontaneously associateswith the polynucleotide complex and be “anchored” thereto through thehydrophobic group. These targeting adducts will typically comprise about1% or less of the polymers in a final composition. In the targetingmolecule, the hydrophobic group can be, among other things, a lipidgroup such as a fatty acyl group. Alternatively, it can be an ionic ornonionic homopolymer, copolymer, block copolymer, graft copolymer,dendrimer or another natural or synthetic polymer.

[0110] In the targeting molecule, the hydrophobic group can be, amongother things, a lipid group such as a fatty acyl group. Alternately, itcan be a block copolymer or another natural synthetic polymer. Thetargeting group of the targeting molecule will frequently comprise anantibody, typically with specificity for a certain cell surface antigen.It could also be, for instance, a hormone having a specific interactionwith a cell surface receptor, or a drug having a cell surface receptor.For example, glycolipids could serve to target a polysaccharidereceptor. It should be noted that the targeting molecule can be attachedto any of the polymer segments identified herein, including R-typepolymeric segments and to the polycationic polymers. For instance, thetargeting molecule can be covalently attached to the free-terminalgroups of the polyether segment of the block copolymer of the invention.Such targeting molecules can be covalently attached to the —OH end groupof the polymers of the formulas XVIII, XIX, XX, and XXI, and the —NH₂end group of the polymers of formulas XVIII (preferably the ε-aminogroup of the terminal lysyl residue), XX or XXIII, or the —COOH endgroup of the polymers of formulas XVIII and XIX. Targeting molecules canbe used to facilitate intracellular transport of the polynucleotidecomposition, for instance transport to the nucleus, by using, forexample, fusogenic peptides as targeting molecules described bySoukchareun et al., Bioconjugate Chem., 6, 43, 1995 or Arar et al.,Bioconjugate Chem., 6, 43, 1995, caryotypic peptides, or otherbiospecific groups providing site-directed transport into a cell (inparticular, exit from endosomic compartments into cytoplasm, or deliveryto the nucleus).

[0111] The polynucleotide component of the compositions of the inventioncan be any polynucleotide, but preferably a polynucleotide with at leastabout 3 bases, more preferably at least about 5 bases. Still morepreferred are at least 10 bases. Included among the suitablepolynucleotides are viral genomes and viruses (including the lipid orprotein viral coat). This includes viral vectors including, but notlimited to, retroviruses, adenoviruses, herpes-virus, and Pox-viruses.Other suitable viral vectors for use with the present invention will beobvious to those skilled in the art. The terms “poly(nucleic acid)” and“polynucleotide” are used interchangeably herein. An oligonucleotide isa polynucleotide, as are DNA and RNA.

[0112] A polynucleotide derivative is a polynucleotide having one ormore moieties (i) wherein the moieties are cleaved, inactivated orotherwise transformed so that the resulting material can function as apolynucleotide, or (ii) wherein the moiety does not prevent thederivative from functioning as a polynucleotide.

[0113] For polyethylene oxide-polypropylene oxide copolymers, thehydrophilic/hydrophobic properties, and micelle formning properties of ablock copolymer are, to a certain degree, related to the value of theratio, n. The ratio, n, is defined as:

n=(|B|/|A|)×(b/a)=(|B|/|A|)×1.32

[0114] where |B| and |A| are the number of repeating units in thehydrophobic and hydrophilic segments of the copolymer, respectively, andb and a are the molecular weights for the respective repeating units.The value of n will typically be between about 0.2 and about 9.0, morepreferably, between about 0.2 and about 1.5. Where mixtures of blockcopolymers are used, n will be the weighted average of n for eachcontributing copolymers, with the averaging based on the weight portionsof the component copolymers. When copolymers other than polyethyleneoxide-polypropylene oxide copolymers are used, similar approaches can bedeveloped to relate the hydrophobic/hydrophilic properties of one memberof the class of polymers to the properties of another member of theclass.

[0115] Surfactant-Containing Polynucleotide Compositions

[0116] The invention also includes compositions of polynucleotide,cationic copolymer, and a suitable surfactant. The surfactant, should be(i) cationic (including those used in various transfection cocktails),(ii) nonionic (e.g., Pluronic or Tetronic), or (iii) zwitterionic(including betains and phospholipids). These surfactants increasesolubility of the complex and increase biological activity of thecompositions.

[0117] Cationic surfactants include but are not limited to primaryamines, secondary amines, tertiary amines (e.g.,N,N′,N′-polyoxyethylene(10)-N-tallow-1,3-diaminopropane), quaternaryamine salts (e.g., dodecyltrimethylammonium bromide,hexadecyltrimethylammonium bromide, mixed alkyl-trimethylammoniumbromide, tetradecyltrimethylammonium bromide, benzalkonium chloride,benzethonium chloride, benzyldimethyldodecylammonium chloride,benzyl-dimethylhexadecylammonium chloride, benzyltrimethylammoniummethoxide, cetyldimethylethylammonium bromide, dimethyldioctadecylammonium bromide, methylbenzethonium chloride, decamethonium chloride,methyl mixed trialkyl ammonium chloride, methyl trioctylammoniumchloride),N,N-dimethyl-N-[2-(2-methyl-4-(1,1,3,3-tetramethylbutyl)-phenoxy]ethoxy)ethyl]-benzenemethanaminiumchloride (DEBDA), dialkyldimetylammonium salts,N-[1-(2,3-dioleyloxy)-propyl]-N,N,N,-trimethylammonium chloride,1,2-diacyl-3-(trimethylammonio)propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl),1,2-diacyl-3-(dimethylammonio)propane (acyl group=dimyristoyl,dipalmitoyl, distearoyl, dioleoyl), 1,2-dioleoyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol, 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester,cholesteryl (4′- trimethylammonio) butanoate), N-alkyl pyridinium salts(e.g. cetylpyridinium bromide and cetylpyridinium chloride),N-alkylpiperidinium salts, dicationic bolaform electrolytes (C₁₂Me₆;C₁₂Bu₆), dialkylglycetyl-phosphorylcholine, lysolecithin, L-α dioleoylphosphatidylethanolamine), cholesterol hemisuccinate choline ester,lipopolyamines (e.g., dioctadecylamidoglycylspermine (DOGS), dipalmitoylphosphatidylethanolamidospermine (DPPES), lipopoly-L(or D)-lysine (LPLL,LPDL), poly(L (or D)-lysine conjugated toN-glutarylphosphatidylethanolamine, didodecyl glutamate ester withpendant amino group (C₁₂GluPhC_(n)N⁺), ditetradecyl glutamate ester withpendant amino group (C₁₄GluC_(n)N⁺), cationic derivatives of cholesterol(e.g., cholesteryl-3β-oxy-succinamidoethylenetrimethylammonium salt,cholesteryl-3β-oxysuccinamidoethylenedimethyl-amine,cholesteryl-3β-carboxyamidoethylenetrimethylammonium salt,cholesteryl-3β-carboxy-amidoethylenedimethylamine,3β[N-(N′,N′-dimethylaminoetane-carbomoil]cholesterol).

[0118] Non-ionic surfactants include but are not limited ton-Alkylphenyl polyoxyethylene ether, n-alkyl polyoxyethylene ethers(e.g., Tritons™), sorbitan esters (e.g., Spans™), polyglycol ethersurfactants (Tergitol™), polyoxyethylenesorbitan (e.g., Tweens™),polysorbates, poly-oxyethylated glycol monoethers (e.g., Brij™,polyoxylethylene 9 lauryl ether, polyoxylethylene 10 ether,polyoxylethylene 10 tridecyl ether), lubrol, copolymers of ethyleneoxide and propylene oxide (e.g., Pluronic™, Pluronic R™, Tetronic™,Pluradot™), alkyl aryl polyether alcohol (Tyloxapol™), perfluoroalkylpolyoxylated amides, N,N-bis[3-D-gluconamidopropyl]cholamide,decanoyl-N-methylglucamide, n-decyl α-D-glucopyranozide, n-decylβ-D-glucopyranozide, n-decyl β-D-maltopyranozide, n-dodecylβ-D-glucopyranozide, n-undecyl β-D-glucopyranozide, n-heptylβ-D-glucopyranozide, n-heptyl β-D-thioglucopyranozide, n-hexylβ-D-glucopyranozide, n-nonanoyl β-D-glucopyranozide1-monooleyl-rac-glycerol, nonanoyl-N-methylglucamide, n-dodecylα-D-maltoside, n-dodecyl β-D-maltoside,N,N-bis[3-gluconamidepropyl]-deoxycholamide, diethylene glycolmonopentyl ether, digitonin, heptanoyl-N-methylglucamide,heptanoyl-N-methylglucamide, octanoyl-N-methylglucamide, n-octylβ-D-glucopyranozide, n-octyl α-D-glucopyranozide, n-octylβ-D-thiogalactopyranozide, n-octyl β-D-thioglucopyranozide.

[0119] Zwitterionic surfactants include but are not limited to betaine(R₁R₂R₃N⁺R′CO₂ ⁻, where R₁R₂R₃R′ are hydrocarbon chains and R₁ is thelongest one), sulfobetaine (R₁R₂R₃N⁺R′SO₃ ⁻), phospholipids (e.g.,dialkyl phosphatidylcholine),3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate,3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate,N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,N-dodecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate,N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,N-octadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate,N-octyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate,N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and dialkylphosphatidyl-ethanolamine.

[0120] The polynucleotide compositions of the invention can beadministered orally, topically, rectally, vaginally, by pulmonary routeby use of an aerosol, or parenterally, i. e. intramuscularly,subcutaneously, intraperitoneallly or intravenously. The polynucleotidecompositions can be administered alone, or it can be combined with apharmaceutically-acceptable carrier or excipient according to standardpharmaceutical practice. For the oral mode of administration, thepolynucleotide compositions can be used in the form of tablets,capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutionsand suspensions, and the like. In the case of tablets, carriers that canbe used include lactose, sodium citrate and salts of phosphoric acid.Various disintegrants such as starch, and lubricating agents such asmagnesium stearate, sodium lauryl sulfate and talc, are commonly used intablets. For oral administration in capsule form, useful diluents arelactose and high molecular weight polyethylene glycols. When aqueoussuspensions are required for oral use, the polynucleotide compositionscan be combined with emulsifying and suspending agents. If desired,certain sweetening and/or flavoring agents can be added. For parenteraladministration, sterile solutions of the conjugate are usually prepared,and the pH of the solutions are suitably adjusted and buffered. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic. For ocular administration, ointmentsor droppable liquids may be delivered by ocular delivery systems knownto the art such as applicators or eye droppers. Such compositions caninclude mucomimetics such as hyaluronic acid, chondroitin sulfate,hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives suchas sorbic acid, EDTA or benzylchronium chloride, and the usualquantities of diluents and/or carriers. For pulmonary administration,diluents and/or carriers are selected to be appropriate to allow theformation of an aerosol.

[0121] The following examples will serve to further typify the nature ofthe invention but should not be construed as a limitation on the scopethereof, which is defined solely by the appended claims.

EXAMPLE 1 Transfection Efficiencies—First Embodiment Complex

[0122] This experiment sought to introduce plasmid pβ-Gal into NIH 3T3cells, a mouse mammary tumor cell line. Plasmid pβ-Gal comprises plasmidpUC19 (available from the Institute of Gene Biology, Russian Academy ofSciences) into which a hybrid of a eukaryotic transcription unit and aE. coli β-galactosidase has been incorporated. With this plasmid, theefficiency of cell uptake can be measured by measuring β-galactosidaseactivity extractable from the treated cells. The copolymer utilized wasa triblock copolymer of formula (XIV) wherein x plus z was 51 and y was39 (hereinafter “Pluronic A”). The polycation utilized waspoly(N-ethyl-4-vinylpyridinium bromide) (“pEVP-Br”). A 10, μg/mlsolution of pβ-Gal (predominantly supercoiled) was prepared in asolution of PBS containing 10 mg/ml of pluronic A and 45, μg/ml ofpEVP-Br. These amounts were calculated to provide a ratio of polycationbasic groups to plasmid phosphate groups of about 10. The ratio ofpluronic A to DNA was about 10⁴. This stock preparation was filtersterilized and a portion was diluted ten fold with serum-free Dulbecco'sModified Eagle's Medium (“DMEM”), so that the concentration of pβ-Galwas 1 μg/ml. This solution was the “Pluronic A transfecting medium.”

[0123] The NIH 3T3 cells were grown in monolayer culture at 37° C. under5% CO₂, utilizing a DMEM medium containing 2 mM glutamine and 10% fetalcalf serum (“FCS”). Cells were grown in monolayer culture were scrapedand prepared for the transaction process by washing three times withfresh medium.

[0124] Aliquots of washed cells that were to be transformed by themethod of the invention were suspended at a concentration of 10⁶cells/ml in Pluronic A transfecting medium. The suspended cells wereincubated for 2 hours at 37° C. and under 5% CO₂. The cells were thenwashed with fresh medium and re-plated.

[0125] Aliquots of cells that were to be transfected by calciumphosphate precipitation were transfected as recommended by Promega ofMadison, Wis., in their manuscript Profection Mammalian TransfectionSystems, Technical Manual, 1990. Specifically, pβ-Gal was mixed with0.25M CaCl₂ The mixture was mixed with an equal volume of 2× HBS (HanksBuffer Salt, available from GIBCO, Grand Island, N.Y.) to create amixture containing 1 μg/mL pβ-Gal. The opaque mixture was incubated atroom temperature for 10 minutes and then applied to the cells. Thesuspended cells were incubated for 2 hours at 37° C. and under 5% CO₂.The cells were then washed with fresh medium and re-plated.

[0126] The repeated cells were incubated for 48 hours in DMEM mediumcontaining 10% FCS. During the incubation, the medium was replaced withfresh medium at 16 hours. After the 48 hour incubation, the cells foreach incubation were collected by scrapping, washed with PBS, andresuspended in 100 μl of 0.2 M Tris-HCL (pH 7.4). The cells were lysedwith several freeze/thaw cycles, and centrifuged at an excess of6,000×/g. 50 μl of supematant was removed from each lysate tube andmixed with 50 μl of a solution of 0.1 mM4-methyl-umbelliferril-β-D-galactopiraniside (the substrate), 0.1 Msodium phosphate (pH 7.4). Each mixture was incubated for 20 min. at 37°C. to allow any 9-galactosidase present to act on the substrate. 50 μlof 0.4 M glycine, pH 10.5, was added to terminate the β-galactosidasereaction. β-galactosidase activity was indicated by the presence ofmethylbelliferon, which can be measured by fluorescence spectroscopy(λ_(ex)=365 nm, λ=450 nm). The results were as follows: Relative EnzymeTreatment Activity ± SEM (n = 4) Pluronic A 320 ± 42 Calcium Phosphate17 ± 5 Precipitation

EXAMPLE 2 Transfection Efficiencies—First Embodiment Complex

[0127] In these experiments, transfection efficiencies with MDCK cells(derived from canine kidney) were examined. Again, pβ-Gal was theindicator polynucleotide. The polycation component of the polynucleotidecomprised a copolymer of N-ethyl-4-vinylpyridinium bromide andN-cetyl-4-vinylpyridiniumn bromide, the monomers incorporated in a molarratio of 97:3, respectively (hereinafter “pEVP-co-pCVP-Br”). The blockcopolymer comprised a triblock copolymer of formula (XIV) wherein x+zwas 18 and y was 23 (hereinafter “Pluronic B”). A Pluronic Btransfecting solution of 1 μg/ml pβ-Gal, 3 μg/ml PEVPco-pCVP-Br, and 1%(w/v) Pluronic B was prepared in Example 1. The ratio of polycationbasic groups to nucleotide Phosphates was about 7. The weight ratio ofPluronic B to pβ-Gal was about 5×10³.

[0128] MDCK cells were plated at 8-10⁵ cells per plate onto 90 mm platesand incubated overnight under serum-containing growth medium. The serumcontaining medium was then replaced with serum-free medium, and thecells were incubated at 37° C., under 5% CO² for 24 hours. For the cellsto be treated with polynucleotide complex, the medium was then replacedwith 5 ml Pluronic B transfecting solution. The cells were incubated,with gentle rocking, at 37° C., under 5% CO₂ In control experiments,cells were transfected with polynucleotide complex, the medium was thenreplaced with 5 ml Pluronic B transfecting solution. The cells wereincubated, with gentle rocking, at 37° C., under 5% CO₂, for 2 hours. Incontrol experiments, cells were transfected using the calcium phosphateprocedure as described above (except that plated cells, not suspendedcells, were transfected).

[0129] After treatment with Pluronic B transfecting solution or calciumphosphate, the cells were washed 5-6 times with fresh medium. They werethen incubated in DMEM containing 10% FCS for 48 hours at 37° C., under5% CO₂. After the first 16 hours of this incubation, the medium wasreplaced. After the incubation, the cells were washed with PBS, releasedfrom their plates by trypsinization, and again washed with PBS.β-Galactosidase was measured as described for Example 1. The resultswere as follows: Relative β-galactosidase Treatment activity ± SEM (n =4) Pluronic B 910 ± 45  Calcium Phosphate 81 ± 17 Precipitation

EXAMPLE 3 Transfection Experiments—First Embodiment Complex

[0130] In these experiments, transfection efficiencies with Chinesehamster ovary (CHO) cells were examined. The polynucleotic component ofthe polynucleotic complex was pβ-Gal. The polycation component comprisedpEVPBr. The block copolymer comprised an octablock copolymer formula(XVII), wherein i was equal to 10 and j was equal to 12 (hereinafter“Pluronic C” available from BASF). A Pluronic C transfecting solution of1 μg/ml pβ-Gal, 4 μg/ml pEVP-Br, and 1% (w/v) Pluronic C was prepared asin Example 1. The ratio of basic groups to nucleotide phosphates was 10.The weight ratio of Pluronic C to pβ-Gal was 10³. The transfectionprotocol was the same as that used in Example 2. The results were asfollows: Relative β-galactosidase Treatment activity ± SEM (n = 4)Pluronic B 910 ± 45  Calcium Phosphate 81 ± 17 Precipitation

EXAMPLE 4 Bacterial Transformation—Second Embodiment Complex

[0131] In these experiments, transformation efficiencies using the MC5strain of Bacillus subtilis were examined. The polynucleotide componentof the polynucleotide complex was plasmid pBC16, a plasmid encodingtetracycline resistance. A block copolymer according to formula (VI) wasused. In particular, the block copolymer was apoly(oxyethylene)-oly((N-ethyl-4-vinylpyridinium bromide) of formula(XXI), wherein i was 44, and j was 20. A stock solution of secondembodiment polynucleotide complex was prepared consistent with thetransfection solutions described above. The ratio of copolymer basicgroups to DNA phosphates in the solution was 0.2. Bacteria weresuspended in Spizizen 11, a transformation media (see, Spizizen,F.N.A.S., U.S.A. 44:1072 (1958)), and aliquots of cells were incubatedin varying concentrations of either polynucleotide complex or freepBC16. The cells were incubated with complex or free DNA for one hour at37° C. Following the incubation, the cells were plated onto agar mediacontaining 10 mg/ml tetracycline. The results, measured by the number oftetracycline-resistant colonies produced under each of the experimentalconditions, were as follows: Transformation (10⁶ clones/ng DNA) DNAPolynucleotide Free concentration (ng/ml) Complex Polynucleotide 5 300(±15) 0 10 450 (±22)  3 (±1) 20 400 (±26)  3 (±4) 50 220 (±17) 20 (±5)

EXAMPLE 5 Protection from Nuclease

[0132] For this example, a complex of plasmid pTZ19 and a diblockcopolymer of formula (XXI)(poly(oxyethylene)-poly((N-ethyl-4vinylpyridinium bromide), wherein iwas 44 and j was 20) was formed. The solution of polynucleotide complexdissolved in PBS contained about 4 μg/ml of plasmid 20 μg/ml of diblockcopolymer. These amounts resulted in a ratio of base groups in thepolycation block to DNA phosphate groups of 5. For control incubations,an equivalent amount of free plasmid was dissolved in buffer. PVUIInuclease was added to solution samples containing free DNA orpolynucleotide complex, and the amount of undigested, circular plasmidDNA, after various digestion times, was determined by electrophoresis ina polyacrylamide gel. See Kabanov et al., Biopolymers, 31:1437-1443(1991). The results were as follows: Circular DNA (% of initial) Time ofIncubation Complex Free DNA 0 100 100 5 100 20 10 100 8 30 100 4 60 1001 180 100 0 600 100 0

EXAMPLE 6 Oligonucleotide Stabilization

[0133] For this example, a complex containing an oligonucleotidecomplementary to the transcription initiation site of the HIV-1 tat gene(“anti-tat”, comprising GGCTCCATTTCTTGCTC) was prepared using thediblock copolymer of formula (XIX)(polyoxyethylene-poly(L-alanine-L-lysine), wherein i is 44 and j is 8).The oligonucleotide complex was prepared in PBS Buffer (pH 7.0) at aconcentration of 0.75 OD₂₆₀/μl oligonucleotide. The ratio of polycationimino and amino groups to polynucleotide phosphate groups was about 50.The mixture was incubated for one hour at room temperature to allow forthe formation of the complex. Then, the complex was purified by gelfiltration chromatography on Sephadex G-25 using 0.05 M NaCl as theeluent. The resulting solution of complex exhibited a concentration of0.11 OD₂₆₀/μl of oligonucleotide. A comparable solution of uncomplexoligonucleotide was prepared. An aliquot of murine blood plasma (10 μl)was mixed with an equal volume of oligonucleotide complex solution or asolution of free oligonucleotide. Samples were incubated at 37° C. forvarious time periods. To stop the reaction of the oligonucleotides withenzymes in the plasma, the samples were diluted with water and extractedwith a water-saturated mixture of phenol:chloroform (1:1). The aqueousphase of the extraction was isolated, and the oligonucleotide thereinwas precipitated with 3% lithium Perchlorate. The precipitate was washedwith acetone, and then dissolved in 100 μl of water. The presence ofundergraded oligonucleotide was determined by high performance liquidchromatography using a C₁₈-Silasorb column (4×90 mm, Gilson, France) anda gradient of acetonitrile in 0.05 M triethylammoniumacetate (pH 7.0) asthe eluent. The results were as follows: Undergraded Time ofoligonucleotide (%) Incubation Complex Free Oligo 0 100 100  3 hours 8828  6 hours 70 17 24 hours 36 0

EXAMPLE 7 Olizonucleotide Stabilization

[0134] This example examined the stability of the oligonucleotidedescribed in Example 6, when complexed with a diblock copolymer offormula (XX) (polyoxyethylene-polypropyleneimine/butyleneimine, whereini is 44 and j is 4-8) was examined. The same methodologies that wereapplied in Example 6 were applied for this example, except that theoligonucleotide concentration was about 0.13 OD₂₆₀/μl. The results wereas follows: Undergraded Time of oligonucleotide (%) Incubation ComplexFree Oligo 0 100 100  3 hours 70 28  6 hours 57 17 24 hours 28 0

EXAMPLE 8 Antisense Cell Incorporation Efficiencies

[0135] This experiment examined the effectiveness of “anti-MDR”, anantisense molecule comprising a 17-chain oligonucleotide of sequenceCCTTCAAGATCCATCCC complementary to positions 422-438 of the mRNAencoding the MDR1 gene product, in reversing multi-drug resistance inSKVLB cells. SKVLB cells are multi-drug resistant cells derived from aovarian cancer cell line. The MDR1gene has been identified asresponsible for the multi-drug resistance in SKVLB cells. Endicott andLing, Ann. Rev. Biochem., 58:137 (1989). In particular, the efficiencyof the anti-MDR oligonucleotide in the polynucleotide complex of theinvention and when in the free state was compared. As controls, the freeand completed form of the anti-tat oligonucleotide described above werealso used. The polynucleotide complexes were formed with the diblockcopolymer of formula (XX)(polyoxyethylene-polypropyleneimine/butyleneimine, where i was 44 and jwas 9-10). The; complexes were prepared by the procedures described inExample 6. The oligonucleotide concentration in the complex or in thefree state was 0.17 OD₂₆₀/μl. The copolymer was present in theconcentration sufficient to define a ratio of polycation segment iminoand amino groups to oligonucleotide phosphate groups of 10.

[0136] The SKVLB cells were incubated for 3 days at 37° C. under 5% CO₂in the presence of free or completed oligonucleotide (at a concentrationof 20 μM based on oligonucleotide content). Fresh media including freeor completed oligonucleotide was added every 12 hours.

[0137] The daunomycin cytotoxicity (IC₅₀) with respect to the cellstreated as described above was measured using the method of Alley et.al., Cancer Res., 48:589-601. The results were as follows: Treatment ofCells Daunomycin IC₅₀ (ng/ml) (n = 4) Control (untreated cells) 8.0Anti-MDR Complex 0.3 Anti-tat Complex 8.2 Free Anti-MDR 2.1 FreeAnti-tat 7.9

EXAMPLE 9 Antisense Oligonucleotide Designed to Inhibit Herpes Virus

[0138] This experiment utilized a 12-chain oligonucleotide, which hadbeen covalently modified at its 5′ end with undecylphosphate substituentand at is 3′ end with a acridine group, was used. This oligonucleotidemodification has been described by Cho-Chung et. al., Biochemistry Int.,25:767-773 (1991). The oligonucleotide sequence utilized, CGTTCCTCCTGU,was complementary to the splicing site at 983-994 of the Herpes SimplexVirus 1 (“HSV-1”). As a control, an equivalently modified sequence(AGCAAAAGCAGG) complementary to the RNA produced by influenza virus wasutilized. The oligonucleotides were applied to HSV-1 infected cells ineither the complexed or the free state. When a complex was utilized, thecomplex was formed with the diblock copolymer of formula (XIX)(polyoxyethylene-poly(L-alanine-L-lysine), wherein i was equal to 44 andj was equal to 8). Oligonucleotide complexes were formed as described inExample 6.

[0139] African marmoset kidney cells (“Vero” cells) were infected withHSV-1 virus (strain L2, obtained from the Museum of Virus Strains, D. I.Ivanovskii, Inst. of Virol., Russian Federation), as described byVinogradov et al., BBRC, 203:959 (1994). The infected cells were washedwith PBS. After washing, fresh RPMI-L 640 media containing 10% of fetalcalf serum and free or complex oligonucleotide was added to the cell.The cells were then incubated at 37° C. under 5% CO₂ for 24 hours. TheHSV-1 infectivity of the of the cell media was then determined using thepatch production method described by Virology, A Practical Approach,Mahy, Ed., IRL Press, Washington, D.C., 1985. The results, utilizingvarying concentrations of oligonucleotide, were as follows: HSV-1Infectious Titre Oligo Conc. (CPE₅₀/ml) (n = 7) Treatment 0.2 μM 1.0 μM5.0 μM Control (un- 1.0 (±0.5) × 10⁶ 1.0 (±0.5) × 10⁶ 1.0 (±0.5) × 10⁶treated infected cells) Anti-HSV 1.4 (±0.2) × 10² 0.5 (±0.3) × 10² 0complex Anti- 1.0 (±0.6) × 10⁶ 0.7 (±0.1) × 10⁶ 0.8 (±0.2) × 10⁶influenza complex Free Anti-HSV 0.9 (±0.4) × 10⁵ 2.3 (±0.7) × 10³ 1.6(±0.4) × 10² Free Anti- 1.1 (±0.4) × 10⁶ 0.9 (±0.2) ± 10⁶ 0.6 (±0.3) ×10⁶ Influenza

EXAMPLE 10 Antisense Oligonucleotide Designed to Inhibit Herpes Virus

[0140] Unless otherwise noted, this example utilized the same proceduresas were utilized in Example 9. The cells utilized were BHK cells, aChinese hamster kidney cell line. When the complexed form of theoligonucleotides was used, the complex was formed with the diblockcopolymer of formula (XVII) (polyoxyethylene-poly-L-lysine, wherein iwas 44 and j was 30), using the procedure described in Example 6. Theconcentration of the stock solution of complex was 0.09 OD₂₆₀/μl. Theratio of polycation segment imino and amino groups to oligonucleotidephosphates was 10. The oligonucleotides, in complexed or free form, wereapplied to the cells at a concentration of 3.0 μM. The results were asfollows: Treatment of cells HSV-1 infectious titre (CPE₅₀/ml) n = 7Control (untreated infected cells)  10 (±3) × 10³ Anti-HSV complex 8(±6) Anti-influenza complex  13 (±4) × 10³ Free Anti-HSV 50 (±14) × 10²Free Anti-influenza  9 (±2) × 10³

EXAMPLE 11 In Vivo Inhibition of HSV

[0141] Polynucleotide complexes between the block copolymer of formula(XVII) (polyoxyethylene-poly-L-lysine, wherein i was 44 and j was 30)and the Anti-HSV and Anti-Influenza oligonucleotides were formed usingthe methods outlined in Example 9. The concentration of the stocksolutions of complexes was 0.9 OD₂₆₀/μl. The ratio of polycation segmentimino and amino groups to oligonucleotide phosphates was 10.

[0142] Inbred white mice (body weight: 6 to 7 g) were infected withHSV-1 (strain Cl from Belorussian Res. Inst. of Epidemiol. & Microbiol.,Minsk) by intraperitoneal injection of 30 μl of a virus suspension(titre: 10⁻⁷ LD₅₀/μl).

[0143] Either Anti-HSV complex, Anti-influenza complex, free Anti-HSV orfree Anti-Influenza were injected (10 μl) into the tail vein of a givenmouse at each of 2, 12, 24, 48 or 72 hours post-infection. The resultswere as follows: Survived animals/ Amount of Animals in a groupTreatment of mice Exp. 1 Exp. 2 Exp. 3 % Survival Control (infectedmice) 1/9 1/10 2/10 13.7 Anti-HSV complex 8/9 6/10 7/10 73.0Anti-influenza complex  2/10 0/10 1/10 10.0 Free Anti-HSV  1/10 1/100/10 7.0 Free Anti-influenza 0/9 1/10 0/10 7.0

EXAMPLE 12 Plasma Life of Polynucleotide Complex

[0144] A ³²P-labelled 17-mer (GGCTCCATTTCTTGCTC) complementary to thetranscription initiation site of the HIV-1 tat gene was utilized in thisexample. The oligonucleotide was modified at its 5′-end with cholesterolas described by Boutorin et al., Bioconjugate Chemistry, 2: 350-356(1990). A polynucleotide conjugate of the oligonucleotide was formedwith the block copolymer of formula (XX)polyoxyethylene-poly(propyleneimine/butyleneimine), wherein i was 44 andj was 9 to 10). The concentration of the stock solution (dissolved inPBS) of complex was 0.18 OD₂₆₀/μl. The ratio of polycation segment iminoand amino groups to oligonucleotide phosphates was 50.

[0145] Male C57/B1/6 mice (weight: 20-24 g; obtained from the RussianResearch Center of Molecular Diagnostics and Therapy, Moscow) received50 μl intravenous injections of Anti-HIV conjugate or free Anti-HIV, at0.18 OD₂₆₀/μl dissolved in PBS. At defined times after the injections,blood sample were taken from the tail vein and the animals weresacrificed. The amount of radioactive material in blood or tissue samplewas determined by liquid scintillation counting (after appropriatesolubilizations). The results were as follows: Plasma levels (% of Timeafter injected dose) Liver levels (% Liver levels (% of injectionAnti-HIV Free Anti- of injected dose) injected dose) (min) Conjugate HIVPrep. A Prep. B 0 100 100 0 0 5 95 58 3 7 10 91 40 5 19 15 84 33 7 26 2079 27 9 30 30 75 20 10 35

EXAMPLE 13 Cationic Block Copolymer Synthesis

[0146] 1,4-dibromobutane (5.4 g, 25 mmoles, from Aldrich Co., Milwaukee,Wis.) was added to a solution of N-(3-aminiopropyl)-1,3-propanediamine(6.55 g, 50 mmoles, from Aldrich Co.) dissolved in 100 ml of1,4-dioxane. This reaction mixture was stirred at 20° C. for 16 h. Theproduct of this reaction spontaneously precipitates from solution as thehydrobromide salt. This precipitated first intermediate was collectedand twice dried by rota-evaporation from a solution of 10% triethylaminein methanol. This evaporation procedure was effective to removesubstantial amounts of the bromide salt. The first intermediate wasdissolved in 50 ml of 1,4-dioxane and reacted with 2.7 g (12.5 mmoles)of 1,4-dibromobutane. Again, the reaction proceeded for 16 h at 20° C.,and the resulting second intermediate was recovered and dried as above.

[0147] The second intermediate was neutralized with acetic acid to a pHof 7-8 and purified by gel filtration on Sephadex G-25, using an aqueouseluent. Three major polymine fractions were obtained, having apparentmolecular weights of 1060, 700 and 500, respectively.

[0148] Poly(oxyethyleneglycol) (1.5 g, M.W. 1500, from Fluka) wasdissolved in 8 ml of 1,4-dioxane and reacted with 0.17 g (1 mmole) ofN,N′-carbonylimidazole (Aldrich Co.) at 20° C. for 3 h. The reactionmixture was divided into two parts. Each part was mixed with 4 ml of a10% (w/v) solution of either the 1060 or 700 MW polyimine fraction,which solution further contained 0.01 N NaOH. The mixture was stirredfor 16 h at 20° C. From this mixture, block copolymers of formula (XX)and various MW ranges were isolated by gel filtration.

EXAMPLE 14 Cationic Block Copolymer Synthesis

[0149] 0.5 g of a succinimidyl carbonate of methoxy-POLY(ETHYLENEGLYCOL) (MW 5000, Shearwater Polymers, Inc., USA) was dissolved in1,4-dioxane. This dioxane solution was added to an aqueous solutioncontaining 0.2 g of the 1060 MW polyimine polymer described above, whichaqueous solution further included

[0150] 0.01 N NaOH. This reaction mixture was stirred at 20° C. for 16h. A polymer of formula (XXII) was isolated from the reaction by gelfiltration.

EXAMPLE 15 Cationic Block Copolymer Synthesis

[0151] 1.5 g of poly(oxyethyleneglylol) (MW 8000, Fluka) were dissolvedin 8 ml of 1,4-dioxane. 0.34 g (2 mmole) of N,N′-carbonylimidazole(Aldrich Co.) were added to the solution and reacted for 3 h at 20° C. 8ml of an aqueous solution containing 0.01 N NaOH and 15% (w/v) of the500 MW polyimine polymer described above in Example 13 was then added tothe first reaction mixture. The resulting mixture was reacted for 16 hat 20° C. with stirring. A polymer of formula (XXIII) was isolated fromthe second reaction mixture by gel filtration.

EXAMPLE 16 Conjugate Synthesis with Oligonucleotide

[0152] A 12-mer oligonucleotide, 5′-CGTTCCTCCTGU (“Oligo A”)complimentary to the splicing site (positions 983-994 on the viralgenome) of the early mRNA of type 1 Herpes Simplex Virus (“HSV-1”), wassynthesized using a 380B-02 DNA-synthesizer (Applied Biosystems, CA).The synthesizer used phosporamidite chemistry and an 8 min. synthesiscycle. Cycle conditions and preparation of the crude product were doneas recommended by Applied Biosystems. The crude Oligo A obtained fromthe synthesis was precipitated from a 1 M LiCl solution (0.5 ml) withacetone (2 ml). The precipitate was dissolved in triethylammoniumacetate buffer and purified by reverse-phase high performance liquidchromatography on a Silasorb C18 column (9×250 mm, Gilson, France)developed with an acetonitrile gradient in a 20 mM TEAA buffer (p H8.5).

[0153] The 3′-terminal of the purified Oligo A was oxidized withperiodate to create an aldehyde and conjugated by reductive alkylationwith a hexamethylene-diamine linker, creating an amine derivative. SeeChe-Chung et al., Biochem. Intemat., 25:767 (1991); Vinogradov et al.,BBRC, 203:959 (1994). “Pluronic A”, a block copolymer of formula(XIV)(x=25, y+38, z=25) was similarly oxidized to create terminalaldehydes. The amine derivative (1 mg) was dissolved in 100 μl of 0.1 Mborate buffer (pH 9.0) and mixed with 2 mg of the Pluronic A derivative.1.5 mg of sodium cyanoborohydride was added to the mixture to reduce theSchiffs bases formed between the amine and aldehyde groups. Thisreaction was allowed to proceed for 12 hours at 4° C. The polymericproduct of this reaction was isolated by gel filtration chromatographyon Sephadex LH-20, utilizing 90% aqueous isopropanol as the eluent. Theconjugate so obtained is referred to hereinafter as “Oligo A Conjugate.”

EXAMPLE 17 The Effect of Oligo A Conjugate on Virus Production

[0154] Oligo A and Oligo A Conjugate were separately dissolved in RPMI1640 medium (ICN Biomedicals Inc., Costa Mesa, Calif.) to a finalconcentration of 0.2 mM (based on oligonucleotide absorbance). Thesestock solutions were then filtered through 0.22 μm filters to remove anypossible bacterial or fungal contamination.

[0155] Monolayers of Vero cells were incubated for 1 hour at 37° C. inserum-free RPMI 1640 together with various concentrations of Oligo A orOligo A Conjugate. The monolayers, while still exposed tooligonucleotides, were then infected with 1 plaque forming unit percultured cell of HSV-1, strain L2 (from the Museum of Virus Strains ofthe D. I. Ivanovskii Institute of Virology, Russian Academy of Sciences,Russian Federation). This infection method has been described byVinogradov et al., BBRC, 203:959 (1994). After 8 hours of exposure tovirus and oligonucleotides, the medium on the cells was replaced withfresh medium containing 10% FCS. Medium from the cells was collected at22 and 39 hours after the ineffective incubation, and the virus titer inthe collected medium was determined as described in Virology, APractical Approach, Mahy, Ed., IRL Press, Oxford Univ. Press,Washington, D.C., 1985. The results were as follows: Infectious Titer ofSample HSV-1 (PFU/ml) concentration Oligonucleotide 22 hours past 39hours past (mM) concentration (μM) infection infection Control (cellswithout 0 5 × 10⁶ 1 × 10⁷ oligonucleotides) Oligo A 10 3 × 10⁶ 5 × 10⁶ 55 × 10⁶ 1 × 10⁷ 2 5 × 10⁶ 1 × 10⁷ 1 5 × 10⁶ 1 × 10⁷ Oligo A Conjugate 100 0 5 0 5 × 10² 2 1 × 10³ 7 × 10³ 1 5 × 10⁴ 3 × 10⁶

EXAMPLE 18 Synthesis of a Phosphonate Monomer

[0156] 40 mmoles of butanediol-1,3 (Merck) dissolved in 50 ml ofanhydrous pyridine (Aldrich) were reacted with 20 mmoles4,4′-dimethoxytritylchloride (Sigma) for 1.5 hours at 20° C. Thereaction was monitored using thin layer chromatography on the silicagelplates (Merck) developed with a chloroform:methanol (95:5). The Rf ofthe product was 0.6. The reaction mixture was added to 200 ml of an 8%aqueous solution of the sodium bicarbonate and the product extractedwith chloroform. The chloroform extract was evaporated in vacuum and theresulting oily first intermediate was used in the next stage of thesynthesis.

[0157] 12 mmoles of first intermediate were dissolved in 30 ml ofanhydrous 1,4-dioxane, containing 3.14 ml (18 mmoles) ofdiisopropylethylamine (Aldrich). 18 mmoles of salicylchlorophosphite(Sigma) dissolved in 10 ml of ahydrous 1,4-dioxane were added to thediisopropylethylamine solution in small portions under an inert, argonatmosphere. The reaction mixture was incubated during 1 hour at 20° C.The reaction was monitored by the thin layer chromatography as describedabove. The Rf of the product was 0.05. 10 mls of water were added to thereaction mixture. After 30 min., the solvent was evaporated. The productwas dissolved in 100 ml of chloroform and the solution obtained waswashed stepwise with (1) 100 ml of 8% aqueous solution of the sodiumbicarbonate, (2) 100 ml of 0.2 M triethyammoniumacelate solution (pH7.2), and (3) 100 ml of water. The organic solvent was evaporated andthe oily remainder, containing the phosphonate monomer was purified bychromatography on silicagel column, using stepwise gradient of (1)chloroform, (2) 3% methanol in chloroform and (3) 6% methanol inchloroform. The yield of the monomer was 4.1 g (=7.3 mmol, 63%). Theproduct, having structure:

[0158] wherein DMT represents a dimethoxytrityl group, can be termed“Phosphonate Monomer A.”

EXAMPLE 19 Synthesis of Polycation BDP

[0159] A 0.05 M solution of the phosphonate Monomer A in anhydrouspyridine:acetonitrile mixture (1:1) was placed in the position 6 of theDNA-synthesator (model 380-B02, Applied Biosystems, CA). A 2% solutionof adamantoilchloride (Sigma) in the mixture acetonitrile:pyridine(95:5) was used as a condensing agent. The synthesis was conducted usingthe program modified for an H-phosphonate cycle (Sinha and Striepeke In:Oligonucleotides and Analogues: A Practical Approach, Eckstein Ed. IRLPress, Oxford, New York-Tokyo, p.185, 1991) and the DMT-group waspreserved after the synthesis was complete. Adenosine (4 μmoles)immobilized on a standard CPG-500 solid support was used as a first unitduring the polymer synthesis (Vinogradov et al. BBRC, 203, 959 (1994).The synthesizer was programmed to add Phosphonate Monomer A repeatingunits to the adenosine monomer. Following all synthesis steps, theH-phosphonate groups on the immobilized substrate were oxidized with thesolution of 104 mg of hexamethylenediamine (Sigma) in 0.6 ml of amixture of anhydrous pyridine:CCl₄ (5:1) applied for 15 min. at 20° C.,then the carrier was washed with the pyridine:acetonitrile mixture(1:1).

[0160] Deblocking and cap removal was achieved by ammonolysis(Oligonucleotides and Analogues. A Practical Approach, Eckstein Ed. IRLPress, Oxford, New York-Tokyo, 1991). The product was purified by HPLCusing Silasorb C,, column (9×250 mm. Gilson, France) in the acetonitrilegradient (0-80%). The peak, containing dimethoxytritylated-product wascollected, the solvent was evaporated and the remainder was treated with80% acetic acid (20 min). The acetic acid was evaporated and thepolycation was purified again by HPLC. The yield of the 15-mer (countedin terms of Phosphonate Monomer A) is 50% (2.2 μmoles). This created apolymer according to formula A. The polymer is termed hereinafter “BDP.”

EXAMPLE 20 Solid Phase Synthesis of the Diblock CopolymerPolyoxyethylene-BDP

[0161] Dimethoxytrityl-polyethyleneoxide-H-phosphonate was synthesizedas described in Example 18 using polyethyleneglycol (1500 M.W. fromFluka) instead of butanediol-1,3. The BDP polycation was synthesized asdescribed in Example 19, except that, at the last stage of the chaingrowth, dimethoxytrityl-polyethyleneoxide-H-phosphonate was introducedas the last building block. The H-phosphonate groups of the blockcopolymer were oxidized as described in Example 19 usingtetramethylenediamine (Sigma) instead of hexamethylenadiamine, resultingin the formation of phosphonamide bonds between the diamines and thebackbone phosphates.

EXAMPLE 21 Solid Phase Synthesis of the Oligonucleotide-BDP DiblockCopolymer.

[0162] A diblock copolymer comprising 12-mer oligonucleotide,5′-GGTTCCTCCTGU (Oligo A, complementary to the splicing site of theearly mRNA of type 1 Herpes Simplex Virus (HSV-1), Vinogradov et al.BBRC, 203, 959 (1994)) and the BDP polymer was synthesized in DNAsynthesator. First the BDP polymer was synthesized as described inExample 19, except that it was not removed from the support. Then theoligonucleotide chain was synthesized step-wise onto BDP polycationicpolymer linked to the solid state support using the standardphosphoroamidite chemistry as described by Vinogradov et al. BBRC, 203,959 (1994). The H-phosphonate groups of the diblock copolymer wereoxidized as described in Example 19 using tetamethylenediamine (Sigma)instead of hexamethylenediamine.

EXAMPLE 22 Effect of Oligonucleotide-BDP Diblock Copolymer on ViralGrowth

[0163] The experiment was performed exactly as described in Example 17except that (1) the oligonucleotide-BDP copolymer of Example 21 was usedand (2) a single concentration of oligonucleotide-BDP copolymer(conjugate) was used (4,4M). Sample Virus titre after 39 hours Control(without oligonucleotide) 500 × 10⁴ Nonmodified Oligo A 500 × 10⁴Diblock  5 × 10⁴

EXAMPLE 23 Synthesis of Branched Polyimine Polycation

[0164] A. The polyimine polycation (“polyspermine”) was obtained bystepwise polycondensation of N-(3-aminopropyl)-1,3-propanediamine and1,4-dibromobutane as described in Example 13 and used withoutconjugating to poly(ethylene glycol).

[0165] B. The polyimine polycation synthesized in A was modified bydansyl chloride to obtain a fluorescent dansyl-labeled substance,purified by thin layer chromatography and a major component of themixture (over 75% in most batches) was analyzed by electrospraymass-spectrometry in positive charge mode. The results were comparedwith mass-spectra obtained for the N-(3-aminopropyl)-1,3-propanediaminemodified with dansyl chloride. Dansyl-labeledN-(3-aminopropyl)-1,3-propanediamine gave a four-modal peak at M+1, M+2,M+3 and M+4 (667.6, 668.5, 669.6 and 670.5). In the spectrum of thepolycondensation products there were observed two types of polymodalpeaks: M and M+54. For M-peaks two distinct groups were observed, withM/2H+ and M/H+, equal to 598.5 and 1195.6 respectively. This molecularmass was very close to a linear polycation with 12 nitrogen atoms(1221). M+54 peaks at 1249.8 and 652.5 correspond to a polycation withCH₂CH₂CH₂CH₂ cross-links.

[0166] C. 1H-NMR spectra were obtained for the samples of the polyiminepolycation synthesized in A and dissolved in DMSO. Three groups ofsignals were observed at 1.40-1.80 ppm (Ha), 1.80-2.20 ppm (Hb) and2.35-2.80 ppm (Hc). Ha related to CH₂CH₂CH₂CH₂ protons, Hb related toCH₂CH₂CH₂ protons, Hc related to —NHCH₂ and protons. Integration ofresonance signals for these three groups gave a ratio Ha:Hb:Hc equal to1.00:0.75:1.20. The theoretical ratio for linear polycations with 12nitrogen atoms is 1.00:1.33:3.67. Increase in Hb:Ha and Hc:Ha ratiossuggested presence of branched structures with a mixture of primary,secondary and tertiary amines.

[0167] D. The concentration of primary amino groups in the polyiminepolycation synthesized in A was determined by fluorescamine method asdescribed by Weigele et al., J. Amer. Chem. Soc., 1972, 94:5927. Thetotal amount of primary, secondary, and tertiary amino groups in thepolycondensation product was determined using potentiometric titration.The ratio of the total amount of primary, secondary, and tertiary aminogroups to the amount primary amino groups equals 2.7. Given themolecular masses of the condensation product determined usingmass-spectrometry the result of this experiment suggests considerablebranching, i.e. the presence of tertiary amines.

EXAMPLE 24 Synthesis of Linear Polyimine Polycation

[0168] Linear polycations of polyimine type are synthesized bycondensation of a diaminoalkyl and bis-aldehyde in the presence ofsodium cyanoborohydride using a modified reductive amination proceduredescribed by Aziz et al., J. Pharmac. Exper. Therapeutics, 1995,274:181. 0.33 g of malonaldehyde bis(dimethyl acetal) was added in 10 mlof 0.5 N HCl and stirred for 1 h at 20° C. to obtain free bis-aldehyde.1.27 g of N,N′-bis[3-aminopropyl]-1,4-butanediamine was added to thissolution and pH was adjusted to 5.0. The mixture was allowed to stay for1 h at 37° C., then 1.27 g of N,N′-bis[3-aminopropyl]-1,4-butanediaminewas added to it and pH was adjusted to 7.0 using sodium carbonatesolution. The reaction mixture was treated with 0.26 g of sodiumcyanoborohydride and left for additional 1 h at 37° C. The finalslightly yellow solution was desalted by gel permeation chromatographyon the Sephadex G-25 column in 10% methanol and first high-molecularweight fractions revealing primary amino groups in ninhydrine test werefreeze-dried. This yields 0.43 g of the following polyimine polycation:

H[NH(CH₂)₃NH(CH₂)₄NH(CH₂)₃]_(x)NH₂

EXAMPLE 25 Synthesis of Cationic Block Copolymer

[0169] 1.5 g of poly(ethylene glycol), methyl ester, mw. 5000 Mw.(Sigma) was activated by 0.25 g of 1,1′-carbonyldiimidazole in 10 ml ofanhydrous acetonitrile for 3 hrs at room temperature. The solvent wasevaporated in vacuo, the residue redissolved in water and dialyzedthrough Membra-Cel MD-25-03.5 membrane with cutoff 3500 Da againstwater. Desalted solution was concentrated in vacuo and used in areaction with 2-fold excess of poly-L-lysine, Mw. 4000, inmethanol-water solution for 16-24 hrs at room temperature. The conjugateobtained was purified by gel-permeation column chromatography onSephadex-50 (fine) (Pharmacia) in water and then by reverse phasechromatography on semi-preparative column (Vydac C18 5 u ,10 mm×25 cm)in acetonitrile concentration gradient. The yield was 70%. Content ofamino groups was measured by fluorescamine method and total nitrogencontent was determined by elemental analysis to assess the purity of theconjugates. Usually it was about 75-90% based on graviometry.

EXAMPLE 26 Synthesis of Cationic Block Copolymer

[0170] Following the procedure of Example 25 but substituting the 2-foldexcess of poly-L-lysine by the same excess of polyethyleneimine,(NHCH₂CH₂)_(x)[N(CH₂CH₂)CH₂CH₂]_(y), Mw. 2000 (Aldrich Co.) there isobtained 0.4 g of the cationic diblock copolymer:

CH₃O(CH₂CH₂O)₁₁₄C(O)(NHCH₂CH₂)_(x)[N(CH₂CH₂)CH₂CH₂]_(y)

EXAMPLE 27 Synthesis of Grafted Copolymer

[0171] A. 24 g (3 mmol) of poly(ethylene glycol), mw 8000 (Aldrich Co.)were dried by co-evaporation with anhydrous pyridine in vacuo anddissolved in 50 ml of anhydrous acetonitrile. Then 0.51 g (1.5 mmol) of4,4′-dimethoxytrityl chloride in 30 ml of anhydrous pyridine was addedto this solution dropwise under continuous stirring during 30 min. Themixture was allowed to stand for additional 2 h at room temperature,then the solvents were evaporated in vacuo. The residue was dissolved in50 ml of dichloromethane, extracted with 5% sodium bicarbonate (2×30ml), and applied on the Silicagel column (3×45 cm, 40-60 μm). Stepwiseelution with dichloromethane-methanol solutions separated a slightlyyellow mono-4,4′-dimethoxytrityl-derivative of poly(ethylene glycol)with an yield about 75-85%. The side product of the reaction (10-15%yield) was the bis-4,4′-dimethoxytrityl-derivative of poly(ethyleneglycol).

[0172] B. 1.5 g of mono-4,4′-dimethoxytrityl-derivative of poly(ethyleneglycol) obtained in A was activated by 0.25 g of1,1′-carbonyldiimidazole in 10 ml of anhydrous acetonitrile for 3 hrs atroom temperature. The solvent was evaporated in vacuo, the residueredissolved in water and dialyzed through Membra-Cel MD-25-03.5 membranewith cutoff 3500 Da against water. Desalted solution was concentrated invacuo and then reacted with poly-L-lysine, Mw. 19000 in methanol-watersolution for 24 h at room temperature at a molar ratio of poly(ethyleneglycol) to free amino groups of poly-L-lysine 0.7:1.0. The conjugateobtained was purified by gel-permeation column chromatography onSephadex-50 (fine) (Pharmacia) in water and then by reverse phasechromatography on semi-preparative column (Vydac C18 5 u ,10 mm×25 cm)in acetonitrile concentration gradient. This yields a grafted polylysinecopolymer at 35% yield in which 50% of free amino groups are substitutedwith poly(ethylene glycol) as determined by fluorescamine method.

EXAMPLE 28 Synthesis of Grafted Copolymer

[0173] A. 24 g (3 mmol) of poly(ethylene glycol), mw 8000 (Aldrich Co.)were dried by co-evaporation with anhydrous pyridine in vacuo anddissolved in 50 ml of anhydrous acetonitrile. Then 0.51 g (1.5 mmol) of4,4′-dimethoxytrityl chloride in 30 ml of anhydrous pyridine was addedto this solution dropwise under continuous stirring during 30 min. Themixture was allowed to stand for additional 2 h at room temperature,then the solvents were evaporated in vacuo. The residue was dissolved in50 ml of dichloromethane, extracted with 5% sodium bicarbonate (2×30ml), and applied on the Silicagel column (3×45 cm, 40-60 μm). Stepwiseelution with dichloromethane-methanol solutions separated a slightlyyellow mono-4,4′-dimethoxytrityl-derivative of poly(ethylene glycol)with an yield about 75-85%. The side product of the reaction (10-15%yield) was the bis-4,4′-dimethoxytrityl-derivative of poly(ethyleneglycol).

[0174] B. 1.5 g of mono-4,4′-dimethoxytrityl-derivative of poly(ethyleneglycol) obtained in A was activated by 0.25 g of1,1′-carbonyldiimidazole in 10 ml of anhydrous acetonitrile for 3 hrs atroom temperature. The solvent was evaporated in vacuo, the residueredissolved in water and dialyzed through Membra-Cel MD-25-03.5 membranewith cutoff 3500 Da against water. Desalted solution was concentrated invacuo and then reacted with polyethyleneimine, Mw. 25,000 inmethanol-water solution for 24 h at room temperature at a molar ratio ofpoly(ethylene glycol) to free amino groups of polyethyleneimine 0.7:1.0.The conjugate obtained was purified by gel-permeation columnchromatography on Sephadex-50 (fine) (Pharmacia) in water and then byreverse phase chromatography on semi-preparative column (Vydac C18 5 μm,10 mm×25 cm) in acetonitrile concentration gradient. This yields agrafted polyethyleneimine block copolymer at 85% in which 45% of freeamino groups are substituted with poly(ethylene glycol) as determined byfluorescamine method as described by Weigele et al. (J. Amer. Chem.Soc., 1972, 94:5927).

EXAMPLE 29 Synthesis of Grafted Copolymer

[0175] Following the procedure of Example 28 but using a molar ratio ofactivated poly(ethylene glycol) to free amino groups ofpolyethyleneimine 0.3:1.0, there is obtained in 80% yield a graftedpolyethyleneimine copolymer in which 24% of free amino groups aresubstituted with poly(ethylene glycol).

EXAMPLE 30 Synthesis of Cationic Block Copolymer

[0176] Following the procedure of Example 26 but substituting 6.0 g ofpolyethyleneglycol, mw 20,000 for the excess of polyethylene glycol, mw5,000 there is obtained 6.0 g of the cationic block copolymer:

CH₃O(CH₂CH₂O)₄₅₆C(O)(NHCH₂CH₂)_(x)[N(CH₂CH₂)CH₂CH₂]_(y)

EXAMPLE 31 Synthesis of Cationic Block Copolymer

[0177] A. Following the procedure of Example 26 but substituting 1.5 gof polyethyleneglycol, Mw. 5,000 by 2.4 g of polyethyleneglycol, Mw.5,000 (Aldrich Co.) there is obtained 1.2 g of the cationic blockcopolymer containing polyethyleneinmine and polyethyleneglycol chainsegments.

[0178] B. The molecular mass of this block-copolymer was determined bystatic light scattering method using DAWN multi-angle laser photometer(Wyatt Technology, Santa Barbara, Calif.) which operated at 15 anglesand equipped with He—Ne laser (632.8 nm). The samples of the blockcopolymer were dialyzed through membrane with cutoff 3,500 Da against4.5×10⁻³ g/ml NaCl and then filtered directly into flow cell used forlight scattering experiments. Weigh-average molecular mass wascalculated on the base of four measurements. Cell constant wasdetermined by calibration with different concentrations of NaCl.Specific refractive index increment (dn/dc) was measured usingWyatt/Optilab 903 interferometric refractometer at 632.8 nm. Themolecular mass of the sample obtained was 16,000, suggesting that thispolymer contained approximately one polyethyleneinmine segment and twopolyethyleneglycol segments.

[0179] C. The number of the primary amino groups in the synthesizedsample of the copolymer was determined using a modified proceduredescribed by Weigele et al. (J.Amer.Chem.Soc., 1972, 94:5927). To 1.5 mlof a sample in 20 mM sodium borate, pH 9.5 (amino groups concentrationup to 100 uM) 0.25 ml of fluorescamine solution (0.024%, Sigma) inacetone was added and vortexed for 5 min. The measurements have beenmade on spectrofluorometer Shimadzu at excitation wavelength 384 nm andat 430 to 510 nm emission wavelength range. Extinction coefficient atemission 475 nm was determined as equal to 1.58×10⁶ M⁻¹. The specificamount of primary amino groups was 0.69 mmol/g.

EXAMPLE 32 Synthesis of Grafted Copolymer

[0180] Following the procedure of Example 28 but substituting 24 g ofpoly(ethylene glycol) by the same amount of Pluronic L61 (BASF Co.) andusing a molar ratio of activated Pluronic L61 to free amino groups ofpolyethyleneimine 0.3:1.0, there is obtained in 22% yield a graftedpolyethyleneimine copolymer in which 8% of free amino groups aresubstituted with Pluronic L61.

EXAMPLE 33 Synthesis of Grafted Copolymer

[0181] Following the procedure of Example 28 but substituting 24 g ofpoly(ethylene glycol), by the same amount of Pluronic P85 and using amolar ratio of activated Pluronic P85 to free amino groups ofpolyethyleneimine 0.3:1.0 there is obtained in 70% yield a graftedpolyethyleneimine copolymer in which 11% of free amino groups ofpolyethyleneimine are substituted with Pluronic P85.

EXAMPLE 34 Synthesis of Grafted Copolymer

[0182] Following the procedure of Example 28 but substituting 24 g ofpoly(ethylene glycol), by the same amount of Pluronic L123 (BASF Co.)and using a molar ratio of activated Pluronic L123 to free amino groupsof polyethyleneimine 0.3:1.0 there is obtained in 30% yield a graftedpolyethyleneimine copolymer in which 9% of free amino groups aresubstituted with Pluronic L123.

EXAMPLE 35 Synthesis of Grafted Copolymer

[0183] Following the procedure of Example 28 but substituting 24 g ofpoly(ethylene glycol), by the same amount of Pluronic F38 (BASF Co.) andusing a molar ratio of activated Pluronic F38 to free amino groups ofpolyethyleneimine 0.3:1.0 there is obtained in 40% yield a graftedpolylysine copolymer in which 9% of free amino groups are substitutedwith Pluronic F38.

EXAMPLE 36 Synthesis of Multi-Grafted Copolymer

[0184] Following the procedure of Example 28 but substitutingpolyethyleneimine by polyethyleneimine modified with Pluronic L123 (BASFCo.) obtained in Example 35 and using a molar ratio of activatedpoly(ethylene glycol) to free amino groups of modified polyethyleneimine0.4:1.0 there is obtained in 20% yield a grafted polyethyleneiminecopolymer in which 9% of free amino groups are substituted with PluronicL123 and 30% of groups are substituted with poly(ethylene glycol).

EXAMPLE 37 Complex with Oligonucleotide

[0185] A. Model phosphorothioate oligodeoxyribonucleotide PS-dT20 wassynthesized using ABI 291 DNA Synthesizer (Applied Biosystems, SanDiego, Calif.) following the standard protocols. After ammoniadeprotection the oligonucleotide was twice precipitated by ethanol andthen used without purification.

[0186] B. The complex formed between the PS-dT20 andpolyethyleneimine-poly(ethylene glycol) block copolymer obtained inExample 28 was obtained by mixing the aqueous solutions of thesepolymers in 10 mM phosphate buffer, pH 7.4 so that the ratio of theprimary amino groups of the block copolymer to the phosphate charges ofthe PS-dT20 was 1.0. All solutions were prepared using double distilledwater and were filtered repeatedly through the Millipore membrane withpore size 0.22 μM.

[0187] C. The electrophoretic mobility (EPM) and the size of theparticles of the complex synthesized in B were determine. The EPMmeasurements were performed at 25° C. with an electrical field strengthof 15-18 V/cm using “ZetaPlus” Zeta Potential Analyzer (BrookhavenInstrument Co.) with 15 mV solid state laser operated at a laserwavelength of 635 nm. The zeta-potential of the particles was calculatedfrom the EPM values using the Smoluchowski equation. Effectivehydrodynamic diameter was measured by photon correlation spectroscopyusing the same instrument equipped with the Multi Angle Option. Thesizing measurements were performed at 25° C. at an angle of 90°. Thezeta potential of this sample was close to zero, suggesting thatparticles were electroneutral. The average diameter of the particles was35 mn.

EXAMPLE 38 Stability Against Nuclease Digestion

[0188] 100 μg of the complex formed between the PS-dT20 andpolyethyleneimine-poly(ethylene glycol) block copolymer obtained inExample 39 was treated by 1 mg of snake venom phosphodiesterase(Phosphodiesterase I from Crotalus adamanteus, 0.024 units/mg, Sigma)for 2 and 18 hrs at 37° C. Reaction mixtures were analyzed by gelpermeation HPLC for digested PS-dT20. The digestion of the PS-dT20 inthis complex was less than 5%. In contrast, free PS-dT20 treated withthe same concentration of enzyme for the same time interval was digestedcompletely.

EXAMPLE 39 Accumulation of Oligonucleotide in Caco-2 Monolayers

[0189] A. A 5′-aminohexyl PS-dT20 oligonucleotide was synthesized usingABI 291 DNA Synthesizer (Applied Biosystems, San Diego, Calif.)following the standard protocols. After ammonia deprotection theoligonucleotide was twice precipitated by ethanol and then used withoutpurification. 5′-Aminohexyl PS-dT20 was labeled by reaction withfluorescein isothiocyanate (Sigma) following the manufacturer protocol.Fluorescein-labeled PS-oligonucleotide was separated from unreactedfluorophore using a Pharmacia PD-10 size exclusion.

[0190] B. The complex formed between the fluorescein-labeled PS-dT20 andpolyethyleneimine-poly(ethylene glycol) block copolymer was synthesizedas described in Example 37 but using fluorescein-labeled PS-dT20 insteadof PS-dT20.

[0191] C. Caco-2 cells, originating from a human colorectal carcinoma(Fogh et al. J. Natl. Cancer Inst., 59:221-226, 1977) were kindlyprovided by Borchardt R. T. (The University of Kansas, Lawrence, Kans.).The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM),containing 10% heat-inactivated fetal bovine serum (FBS), 1%non-essential amino acids, benzylpenicilin (100 U/ml) and streptomycin(10 ug/mi), in an atmosphere of 90% air and 10% CO₂ as described byArtursson (J. Pharm. Sci., 79:476-482, 1990). All tissue culture mediawere obtained from Gibco Life Technologies, Inc. (Grand Island, N.Y.).The cells were grown on collagen coated polycarbonate filter chamberinserts (Transwell, Costar Brand Tissue Culture Products, Contd.; poresize 0.4 um; diameter 24.5 mm). 250,000 cells were added to each insertand cells of passage number 32-45 were used. The cells were fed everysecond day and were allowed to grow and differentiate for up to 14 daysbefore the monolayers were used in the following absorption experiments.

[0192] D. Caco-2 cell monolayers were preincubated for 30 min. at 37° C.with assay buffer, containing sodium chloride (122 mM), sodiumbicarbonate (25 mM), glucose (10 mM), HEPES (10 mM), potassium chloride(3mM), magnesium sulfate (1.2 mM), calcium chloride (1.4 mM) andpotassium phosphate dibasic (0.4 mM). After this, the assay buffer wasremoved and the cells were exposed to 50 μM fluorescein-labeledPS-oligonucleotide or its complex in the assay buffer for 90 min at 37°C. After that the dye solutions were removed and cell monolayers werewashed three times with ice-cold PBS. Cells were then solubilized in1.0% Triton X-100 and aliquots (25 μl) were removed for determination ofcellular fluorescence using a Shimadzu RF5000 spectrofluorometer atλex=488 nm, λem=520 nm. Samples were also taken for proteindetermination using the Pierce BCA method. The amounts offluorescein-labeled PS-dT20 absorbed by the cells was as follows:Cellular accumulation of Sample oligonucleotide, nmol/mg protein Freefluorescein-labeled PS-dT20 0.14 ± 0.03 The complex  0.5 ± 0.01

[0193] This demonstrates that incorporation of polynucleotide in thecomplex with the block copolymer increases cellular accumulation ofpolynucleotide by more than 3-times.

EXAMPLE 40 Transport of Oligonucleotide Across Caco-2 Monolayers

[0194] A. The filter-grown Caco-2 monolayers were used foroligonucleotide permeability studies after complete maturation, i.e., asfrom day 14 after plating. Filters were gently detached from the wellsand placed in Side-Bi-Side diffusion cells from Crown Bio Scientific,Inc. (Somerville, N.J.) maintained at 37° C.±0.1° C. This system is usedas an in vivo model of human intestinal epithelium to evaluate oralbioavailability of drugs (Pauletti et al. Pharm. Res., 14:11-17, 1977).Cell monolayers were preincubated for 30 minutes at 37° C. with theassay buffer, containing 10% heat-inactivated fetal bovine serum (FBS),1% non-essential amino acids, benzylpenicilin (100 U/ml) andstreptomycin (10 ug/ml), added to both donor and receptor chambers (3ml). After preincubation, the assay buffer in the receptor container wasreplaced by the fresh one, while the assay buffer in the donor containerwas replaced by 50 μM fluorescein-labeled PS-oligonucleotide or itscomplex in the assay buffer. To account for the integrity of themonolayers the rhodamine 123 solutions in the donor container alsocontained H³-labeled manitol, a paracellular marker (Dawson, J. MembraneBiol., 77: 213-233, 1977) obtained from DuPont Corp. (Boston, Mass.). At120 min., the solutions in the receptor chamber were removed fordetermination of fluorescein-labeled PS-oligonucleotide using a ShimadzuRF5000 fluorescent spectrophotometer (λex=488 nm, λem=520 nm) andH³-manitol determination using a liquid scintillation counter (HewlettPackard Instruments). Immediately after collecting the solutions in thereceptor chamber 3 ml of fresh assay buffer was added to this chamber.The transport of fluorescein-labeled PS-oligonucleotide (or manitol)across Caco-2 cell monolayers was expressed as a percentage of the totalfluorescein-labeled PS-oligonucleotide (or manitol) accumulated in thereceptor chamber to the initial amounts of fluorescein-labeledPS-oligonucleotide (or manitol) in the donor chamber. A minimum of threedifferent membranes was studied for each drug composition at multipletime points for each membrane. The results were as follows: SamplePS-dT20 transport, % Manitol transport, % Free fluorescein-labeled 0.001± 0.0005 4.0 ± 0.1  PS-dT20 The complex 0.075 ± 0.005  4.2 ± 0.02

[0195] This demonstrates that incorporation of polynucleotide in thecomplex with the block copolymer increases transport of thispolynucleotide across Caco-2 monolayers by more than 7-times while thetransport of paracellular marker is not affected.

EXAMPLE 41 Synthesis of Polyvinylpyrrolidone-Polyethyleneimine Conjugate

[0196] A. Carboxyterminated polyvinylpyrrolidone was obtained using themethod described by Torchilin et al. (J. Pharm. Sci. 84:1049, 1995).Polyvinylpyrrolidone was synthesized by chain transfer free radicalpolymerization of 50% wt. N-vinylpyrrolidone (Sigma) inisopropoxyethanol with 1% wt. 2,2′-azoisobutyronitrile (Sigma) as theinitiator. The MW of the polymer obtained was about 6,000 as determinedby viscosimetry and gel-permeation chromatography on Sephadex G25. Theterminal OH group of polyvinylpyrrolidone was converted into a COOHgroup by activating the OH group with 4-nitrophenyl chlorophormate andsubsequently coupling it with glycine as described by Sartore et al., J.Bioact. Compat. Polym. 9:411 (1994).

[0197] B. Six grams (1 mmole) of carboxyterminated polyvinylpyrrolidoneobtained in Part A dissolved in 30 ml of dry dioxane was treated with0.2 g of N,N′-dicyclohexylcarbodiimide for 3 hours at room temperatureand then reacted with 2.5 g (0.1 mmole) polyethyleneimine, Mw. 25,000(Aldrich Co.) for 15 hours at room temperature. The reaction mixture wasthen dialyzed against water for 3 days using Spectra/Por membranes andthen purified by high-pressure liquid chromatography using Silasorb C18column in the gradient of acetonitrile. Three (3) g ofpolyvinylpyrrolidone-polyethyleneimine conjugate was obtained.

EXAMPLE 42 Synthesis of Polyaryloylmorpholine-PolyethyleneimineConjugate

[0198] A. Carboxyterminated polyaryloylmorpholine was obtained using themethod described by Torchilin et al. (J. Pharm. Sci., 84:1049, 1995).The monomer was synthesized by acylation of morpholine (Aldrich) withacryloyl chloride (Aldrich), which was then polymerized in water with 1%wt. 2,2′-azoisobutyronitrile (Sigma) as the initiator and2-mercaptoacetic acid as chain transfer reagent (Ranucci et al.,Macromol. Chem. Phys., 195:3469, 1994). The molecular weight (MW) of thepolymer obtained was ca. 8,000 as determined by viscosimetry andgel-permeation chromatography on Sephadex G25.

[0199] B. Following the procedure of Example 40 but substituting 16 g ofcarboxyterminated polyaryloylmorpholine for 6 g of carboxyterminatedpolyvinylpyrrolidone in Part B there was obtained 4 g ofpolyaryloylmorpholine-polyethyleneimine conjugate.

EXAMPLE 43 Synthesis of Polyvinylpyrrolidone-Polyethyleneimine Conjugate

[0200] A. Carboxyterminated polyacrylamide was obtained using the methoddescribed by Torchilin et al. (Biochim. Biophys. Acta., 1195:181, 1994).Ten percent by weight of acrylamide (Aldrich) was polymerized in dioxanewith 1% wt. 2,2′-azoisobutyronitrile (Sigma) as initiator and2-mercaptoacetic acid as chain transfer reagent. The MW of the polymerobtained was ca. 5,000 as determined by viscosimetry and gel-permeationchromatography on Sephadex G25.

[0201] B. Following the procedure of Example 40 but substituting 25 g ofcarboxyterminated polyacrylamide for 6 g of carboxyterminatedpolyvinylpyrrolidone in Part B, 10 g ofpolyvinylpyrrolidone-polyethyleneimine conjugate was obtained.

EXAMPLE 44 Synthesis of Polyacrylamide-Polyethyleneimine Conjugate

[0202] Following the procedure of Example 42 but substitutingvinyltriazole monomer for acrylamide monomer in Part A, 9.2 g ofpolyacrylamide-polyethyleneimine conjugate was obtained.

EXAMPLE 45 Synthesis of Polyvinylalcohol-graft-PolyethyleneimineCopolymer

[0203] 20 g of polyvinylalcohol, mw. 100,000 (Aldrich) was activated by2.5 g of 1,1′-carbonyldiimidazole in 30 ml of anhydrous acetonitrile for4 hrs at room temperature. The solvent was evaporated in vacuo, and theresidue redissolved in water and dialyzed through Membra-Cel MD-25-03.5membrane against water. Desalted solution was concentrated in vacuo andused in a reaction with 5 g of polyethyleneinimine, mw. 2000, inmethanol-water solution for 30 hrs at room temperature. The conjugateobtained was purified by gel-permeation column chromatography onSephadex-50 (fine) (Pharmacia). 16.5 g ofpolyvinylalcohol-graft-polyethyleneimine copolymer was obtained.

EXAMPLE 46 Synthesis of Monoamino-Poly(Ethylene Glycol)

[0204] A. Poly(ethylene glycol), MW 8,000, obtained from Aldrich (StLouis, Mo.) was modified by one terminal hydroxyl group using with4,4′-dimethoxytrityl (DMT) chloride (Sigma, St Louis, Mo.). Briefly, 16g (2 mmoles) of PEG were dried by coevaporation in vacuo with anhydrouspyridine (3×50 ml) and dissolved in 100 ml of the solvent. 0.34 g (1mmole) of 4,4′-dimethoxytrityl chloride in 10 ml of anhydrous pyridinewas then added to this solution dropwise for 30 min and the mixture wasleft under stirring overnight at 25° C. Monotritylated polymer waseseparated from fast moving bis-substituted by-products and non-reactedinitial compounds by preparative column chromatography on Silicagel(Selecto Scientific, Norcross, Ga.), particle size 32-63 μm, using astepwise elution by methanol in dichloromethane. Fractions containingmono DMT-substituted poly(ethylene glycol) (DMT-PEG) were collected andsolvents were removed in vacuo. DMT-PEG was obtained with 40% yield.

[0205] B. To prepare the monoamino-poly(ethylene glycol) (N-PEG), 1.5 gof DMT-PEG (0.18 mmol) was coevaporated twice in vacuo with anhydrousacetonitrile (10 ml) and treated by an excess of 1,1′-carbonyin bound 34μg of biotin-poly(ethylene glycol)-polyethylenymer comprising apolyether segment and a polycation segment. In yet another aspect, theinvention provides polynucleotides 10 that have been covalently modifiedat their 5′ or 3′ end to attach a polyether polymer segment.

What is claimed:
 1. A polymer of a plurality of covalently bound polymer segments wherein said segments comprise: (a) at least one polycation segment which is a cationic homopolymer or copolymer comprising at least three cationic amino acids, or at least three aminoalkylene monomers, said monomers being selected from the group consisting of: (i) at least one tertiary amino monomer of the formula:

 and the quaternary salts of said tertiary amino monomer, and (ii) at least one secondary amino monomer of the formula:

 and the acid addition and quaternary salts of said secondary amino monomer, in which: R¹ is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; each of R² and R³, taken independently of the other, is the same or different straight or branched chain alkanediyl group of the formula: —(C_(z)H_(2z))—  in which z has a value of from 2 to 8; R⁴ is hydrogen satisfying one bond of the depicted geminally bonded carbon atom; and R⁵ is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; R⁶ is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; R⁷ is a straight or branched chain alkanediyl group of the formula: —(C_(z)H_(2z))—  in which z has a value of from 2 to 8; and R⁸ is hydrogen, alkyl of 2 to 8 carbon atoms, an A monomer, or a B monomer; and (b) at least one water-soluble nonionic polymer segment.
 2. The polymer according to claim 1 wherein the water-soluble nonionic polymer segment is a homopolymer or copolymer of at least one of the monomers selected from the group consisting of acrylamide, gycerol, vinylalcohol, vinylpyrrolidone, vinylpyridine, vinylpyridine N-oxide, oxazoline, or a acroylmorpholine, and derivatives thereof.
 3. The polymer according to claim 1 wherein the water-soluble nonionic polymer is of the formula:

in which m has a value of from 3 to about 10,000.
 4. The polymer according to claim 1 wherein said water-soluble nonionic polymer segment comprises at least one straight or branched chained polyether segment having from about 5 to about 400 monomeric units which is: (i) a homopolymer of a first alkyleneoxy monomer —OC_(n)H_(2n)— or (ii) a copolymer or block copolymer of said first alkyleneoxy monomer and a second different alkyleneoxy monomer —OC_(m)H_(2m)—, in which n has a value of 2 or 3 and m has a value of from 2 to
 4. 5. A composition comprising the polymer of claim 1 and a polynucleotide.
 6. A composition comprising the polymer of claim 1 and a targeting molecule.
 7. A composition comprising the polymer of claim 1, a polynucleotide and a surfactant.
 8. The composition according to claim 4 wherein said surfactant is cationic, nonionic, or zwitterionic.
 9. A composition comprising the polymer of claim 1 and a virus.
 10. A composition comprising the polymer of claim 1 and at least one of a ligand and a receptor.
 11. A composition comprising the polymer of claim 1 and at least one of biotin, avidin, and streptavidin.
 12. A method of treating a mammal in need of treatment comprising administering to said mammal an effective amount of the polymer of claim
 1. 13. A copolymer comprising at least one polynucleotide segment and at least one polyether segment, said polyether segment comprising oxyethylene and oxypropylene. 